Compositions comprising modified collagen and uses therefor

ABSTRACT

The invention provides modified collagen and related therapeutic and diagnostic methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/630,271, which was filed on Nov. 23, 2004, and U.S. ProvisionalApplication No. 60/722,079, which was filed on Sep. 29, 2005, each ofwhich is hereby incorporated by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by the National Institutes of Health(Grant No. GM-74812) and the National Science Foundation (CTS-0210220).The government may have certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 18, 2011, isnamed 64411US7.txt and is 48,935 bytes in size.

BACKGROUND OF THE INVENTION

Collagen is used in a variety of medical applications ranging fromhemostatic materials and biocompatible coatings to drug delivery andtissue engineering. Collagen-based biomaterials are presently used forsoft-tissue engineering and repair, and medical products composed ofcollagen have been approved by the FDA, and are commercially available.These include collagen-based corneal shields, anti-infectious catheters,tissue sealants, hemostatic sponges, and topical wound dressingproducts. Collagen is also used for tissue engineering substratestargeted for skin, bone, and blood vessel replacement. There isincreasing demand for biocompatible and biofunctional materials thatfeature collagen as a scaffold to support tissue growth, to promotehealing, and to develop engineered tissues for organ replacementtherapies.

Traditionally, collagen is used as a passive biomaterial that protectsinjured sites and supports healing processes. Methods for modifyingcollagen would be useful for the development of materials that canparticipate in tissue regeneration by actively regulating celldifferentiation, proliferation, and tissue organization. Methods forproducing modified collagens have focused on chemical modification, thatinclude coupling modifying groups to collagen's amino acid side chains;however, the chemical reactions between exogenous components andcollagen molecules are toxic and difficult to control because collagenmolecules are large and complex biopolymers. Progress in the area oftissue repair and regeneration requires better methods for conjugatingexogenous functionalities to collagen. Desirably, such methods shouldovercome challenges associated with controlling chemical reactions andchemical toxicity.

SUMMARY OF THE INVENTION

As described below, the present invention features compositionscomprising modified collagen and therapeutic and diagnostic methodsrelated to the use of such compositions.

In one aspect, the invention features a method for modifying collagen,the method involving contacting collagen with a collagen mimeticpeptide, under conditions that provide for a physical interactionbetween the collagen and the collagen mimetic peptide.

In another aspect, the invention provides a modified collagen made bythe process of the above aspect.

In another aspect, the invention features a collagen mimetic peptideconjugate, where the conjugate is selected from the group consisting ofan antibiotic, a cell adhesion molecule, a contrast agent, a detectablelabel, a growth factor, a component of the extracellular matrix, ananti-inflammatory, a polymer, PEG, and a small molecule. In oneembodiment, the collagen mimetic peptide contains a Z-[X-Y-Gly]_(n)repeat unit (SEQ ID NO: 1), where Z is any amino acid, X is proline ormodified proline, Y is proline or modified proline, and n is an integerbetween 1 and 20, inclusive.

In a related aspect, the invention provides collagen mimetic peptidethat contains amino acid sequences Gly_(n)-(ProHypGly)_(n′) (SEQ ID NO:2), (ProHypGly)_(n′)-Tyr_(n) (SEQ ID NO: 3), orCys_(n)-(Pro-Hyp-Gly)_(n′) (SEQ ID NO: 4) where n is an integer between1 and 5, and n′ is any integer between 1 and 15, and the peptide furthercomprises a modifier selected from the group consisting of anantibiotic, a cell adhesion molecule, a contrast agent, a detectablelabel, a growth factor, an anti-inflammatory, a component of theextracellular matrix, a polymer, PEG, and a small molecule. In oneembodiment, n′ is 5, 6, 7, or 8.

In another aspect, the invention provides a modified collagen containinga CMP mimetic peptide of any previous aspect or made by the process of aprevious aspect. In yet another aspect, the invention provides a methodof modifying the adhesiveness of a composition, the method involvingcontacting the composition with a CMP conjugate. In one embodiment, thecomposition comprises collagen, for example, in a three-dimensionalmatrix or in a collagen film. In other embodiments, the collagen ismodified in a micropattern. In one embodiment, the CMP conjugate is apoly(ethyleneglycol)/CMP conjugate. In another embodiment, modificationincreases or decreases adhesiveness.

In another aspect, the invention provides a nanoparticle containing acollagen mimetic peptide fixed to the nanoparticle. In one embodiment,the collagen mimetic peptide contains the following sequenceCys_(n)-(Pro-Hyp-Gly)_(n′) (SEQ ID NO: 5), where n is any integerbetween 1 and 5, and n′ is any integer between 1 and 10. In anotherembodiment, n is 1 and n′ is 3. In another embodiment, n′ is 5, 6, 7, or8. In yet another embodiment, the collagen mimetic peptide is fixed tothe surface of the nanoparticle. In another embodiment, the nanoparticleis between 5 and 20 nm (e.g., between 10 and 15 nm in diameter). Inanother embodiment, the nanoparticle is a metal, such as gold (AU).

In another aspect, the transmission electron microscopy marker containsthe nanoparticle of any previous aspect. In another embodiment, thenanoparticle comprises AU.

In another aspect, the invention provides a diagnostic marker containinga detectable CMP conjugate, where the marker binds collagen and detectsa disease. In one embodiment, CMP is conjugated to a nanoparticle, adetectable label, or a contrast reagent. In another embodiment, themarker detects a disease characterized by disruption of collagenstructure. In another embodiment, the marker detects the disease bybinding to a collagen selected from the group consisting of collagentype 1-type 29 (e.g., type I, II, III, IV, IX, X, or XI). In oneembodiment, the disease is selected from the group consisting ofEhlers-Danlos syndrome, osteogenesis imperfecta, achondrogenesis type 2,hypochondrogenesis, Kniest dysplasia, otospondylomegaepiphysealdysplasia, spondyloepimetaphyseal dysplasia, Strudwick type,spondyloepiphyseal dysplasia congenita spondyloperipheral dysplasia,Stickler syndrome, and Weissenbacher-Zweymüller. In one embodiment, themarker is detectable using transmission electron microscopy.

In another aspect, the invention provides a method for diagnosing asubject as having or having a propensity to develop a diseasecharacterized by disruption of a collagen structure, the methodinvolving contacting a patient sample with a detectable CMP-conjugate;and detecting an alteration in a collagen structure present in thepatient sample, where the alteration diagnoses the subject as having orhaving a propensity to develop a disease characterized by disruption ofa collagen structure.

In another aspect, the invention provides a method for diagnosing asubject as having or having a propensity to develop a thrombosis, themethod involving contacting a blood vessel with a detectableCMP-conjugate; and detecting an alteration present in the blood vessel,where the alteration diagnoses the subject as having or having apropensity to develop a thrombosis. In one embodiment, the detectableCMP-conjugate binds a collagen selected from the group consisting ofcollagen type 1-type 29 (e.g., type I, II, III, IV, IX, X, or XI). Inanother embodiment, the disease is selected from the group consisting ofEhlers-Danlos syndrome, Osteogenesis imperfecta, achondrogenesis type 2,hypochondrogenesis, Kniest dysplasia, otospondylomegaepiphysealdysplasia, spondyloepimetaphyseal dysplasia, Strudwick type,spondyloepiphyseal dysplasia congenita spondyloperipheral dysplasia,Stickler syndrome, and Weissenbacher-Zweymüller. In another embodiment,the marker binds a collagen present in a thombosis or a vessel wall.

In another aspect, the invention provides diagnostic kit containing adetectable CMP conjugate and directions for the use of the conjugate inthe diagnosis of a disease characterized by a disruption in collagenstructure. In one embodiment, the detectable CMP-conjugate binds acollagen selected from the group consisting of collagen type 1-type 29(e.g., type I, II, III, IX, X, or XI). In another embodiment, thedisease is selected from the group consisting of Ehlers-Danlos syndrome,Osteogenesis imperfecta, achondrogenesis type 2, hypochondrogenesis,Kniest dysplasia, otospondylomegaepiphyseal dysplasia,spondyloepimetaphyseal dysplasia, Strudwick type, spondyloepiphysealdysplasia congenita spondyloperipheral dysplasia, Stickler syndrome, andWeissenbacher-Zweymüller.

In another aspect, the invention provides a composition for repellingcell adhesion, the composition containing a CMP-poly(ethylene glycol)conjugate. In one embodiment, CMP-poly(ethylene glycol) conjugatecomprises methoxy-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6) or(Pro-Hyp-Gly)₉-Gly₃-PEG₅₀₀₀ (SEQ ID NO: 7). In another embodiment,CMP-poly(ethylene glycol) conjugate comprises grafted linear PEG or NHSactivated star-shaped PEG. In another embodiment, the compositioncomprises star shaped PEG having the following

formula:In another embodiment, the composition comprisesAcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 8) In another embodiment,the CMP comprises -(Pro-Hyp-Gly)_(n′) (SEQ ID NO: 9), where n is anyinteger between 1 and 3, and n′ is any integer between 1 and 10.

In another aspect, the invention provides matrix containing aCMP-polymer conjugate, where the CMP-polymer conjugate alters theadhesive properties of the matrix. In one embodiment, theCMP-poly(ethylene glycol) conjugate comprisesmethoxy-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6) or(Pro-Hyp-Gly)₉-Gly₃-PEG₅₀₀₀ (SEQ ID NO: 7). In another embodiment, theCMP-poly(ethylene glycol) conjugate comprises grafted linear PEG. Inanother embodiment, the composition comprises NHS activated star-shapedPEG. In another embodiment, the composition comprises star shaped PEGstar shaped PEG having the following formula:

In another embodiment, the composition comprisesAcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 8). In anotherembodiment, the matrix further contains a cell selected from the groupconsisting of chondrocytes, endothelial cells, dendritic cells, stemcells, multipotent progenitor cells, skin cells, liver cells, heartcells, kidney cells, pancreatic cells, lung cells, bladder cells,stomach cells, intestinal cells, cells of the urogenital tract, breastcells, skeletal muscle cells, skin cells, bone cells, cartilage cells,keratinocytes, hepatocytes, gastro-intestinal cells, epithelial cells,endothelial cells, mammary cells, skeletal muscle cells, smooth musclecells, parenchymal cells, and osteoclasts. In another embodiment, theCMP-poly(ethylene glycol) conjugate is present in a pattern. In yetanother embodiment, the CMP comprises -(Pro-Hyp-Gly)_(n′) (SEQ ID NO:9), where n is any integer between 1 and 3, and n′ is any integerbetween 1 and 10.

In another aspect, the invention provides method for repelling celladhesion on a composition, the method involving fixing a CMP-PEGconjugate to a composition, where the CMP-PEG conjugate repels celladhesion on the composition. In one embodiment, the CMP-poly(ethyleneglycol) conjugate comprises methoxy-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ IDNO: 6) or (Pro-Hyp-Gly)₉-Gly₃-PEG₅₀₀₀ (SEQ ID NO: 7). In anotherembodiment, the CMP-poly(ethylene glycol) conjugate comprises graftedlinear PEG or NHS activated star-shaped PEG. In another embodiment, themethod repels adhesion of a cell selected from the group consisting ofendothelial cells, dendritic cells, stem cells or other multipotentprogenitor cells, skin cells, liver cells, heart cells, kidney cells,pancreatic cells, lung cells, bladder cells, stomach cells, intestinalcells, cells of the urogenital tract, breast cells, skeletal musclecells, skin cells, bone cells, cartilage cells, keratinocytes,hepatocytes, gastro-intestinal cells, epithelial cells, endothelialcells, mammary cells, skeletal muscle cells, smooth muscle cells,parenchymal cells, osteoclasts, and chondrocytes.

In another aspect, the invention provides composition containing aCMP-poly(ethylene glycol) conjugate that comprises a detectable marker.In one embodiment, CMP-poly(ethylene glycol) conjugate comprisesmethoxy-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6) or(Pro-Hyp-Gly)₉-Gly₃-PEG₅₀₀₀ (SEQ ID NO: 7). In another embodiment, theCMP-poly(ethylene glycol) conjugate comprises grafted linear PEG, NHSactivated star-shaped PEG, or star shaped PEG having the formula shownabove.

In yet another embodiment, the composition comprisesAcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 8).

In another aspect, the invention provides a method of determining thepresence of a CMP-poly(ethylene glycol) conjugate in a composition, themethod involving contacting a composition with a CMP-poly(ethyleneglycol) conjugate containing a detectable marker; and detecting bindingof the CMP-poly(ethylene glycol) conjugate to the composition. In oneembodiment, the detecting quantifies the level of detectable marker. Inanother embodiment, the detecting localizes the detectable marker in thecomposition.

In another aspect, the invention provides a composition for crosslinkingcollagen, the composition containing a CMP-poly(ethylene glycol)conjugate. In one embodiment, CMP is conjugated to a multi-armed PEGcompound. In another embodiment, PEG comprises an amino group, a thiolgroup. In one embodiment, the composition comprises star shaped PEG of aFormula shown above. In another embodiment, the composition comprisesAcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 8).

In another aspect, the invention provides method for preventing ortreating thrombosis in a subject, the method involving contacting ablood vessel having or having a propensity to develop a thrombosis witha CMP-anti-thrombosis conjugate; where the contacting prevents or treatsa thrombosis. In one embodiment, the propensity to develop a thrombosisis related to angioplasty. In another aspect, the invention provides thevessel is contacted with a hydrogel containing a CMP-anti-thrombosisconjugate. In another embodiment, the CMP-anti-thrombosis conjugatebinds collagen present in the thrombosis. In another embodiment, theCMP-anti-thrombosis conjugate degrades the thrombosis. In anotherembodiment, the method further involves monitoring the efficacy of themethod (e.g., by determining plasma level of β-thromboglobulin orthrombin-antithrombin complexes).

In yet another aspect, the invention provides diagnostic marker thatdetects a vessel having or having an increased propensity to develop athrombosis relative to a control vessel, the marker containing adetectable CMP conjugate that binds collagen. In one embodiment, themarker detects the presence of a thrombosis in the vessel. In anotherembodiment, the marker binds collagen type III. In yet anotherembodiment, the marker detects a vessel having a propensity to develop athrombosis.

In another aspect, the invention provides a diagnostic kit containingthe diagnostic marker of any previous aspect and directions for the useof the conjugate in the diagnosis of a thromobosis or a propensity todevelop a thrombosis.

In another aspect, the invention provides an implantable, sustainedrelease device containing a CMP conjugate fixed to a collagen matrix. Inone embodiment, the conjugate is selected from the group consisting ofan antibiotic, a growth factor, an anti-inflammatory, a component of theextracellular matrix, and a small molecule. In another embodiment, theantibiotic is selected from the group consisting of penicillin,tetracycline, plectasin, LAH4. In another embodiment, the growth factoris selected from the group consisting of angiogenin, erythropoietin,vascular endothelial growth factor (VEGF), granulocyte/macrophage colonystimulating factor, macrophage-colony stimulating factor,platelet-derived endothelial cell growth factor, and platelet-derivedgrowth factor. In another embodiment, the small molecule is selectedfrom the group consisting of anti-thrombotics, anti-atherosclerosisagents, cartilage repair agent. In one embodiment, the collagen matrixis a hydrogel. In another embodiment, the collagen matrix is a pellet ora film.

In another aspect, the invention provides a kit containing an effectiveamount of an implantable, sustained release device of a previous aspect,and directions for the use of the device in the treatment of a diseaseor disorder.

In another aspect, the invention provides a hemostatic sponge containinga collagen mimetic peptide and collagen. In one embodiment, the collagenmimetic peptide physically associates with the collagen. In anotherembodiment, the sponge further contains a natural or synthetic polymer.In another embodiment, the collagen mimetic peptide further comprises aconjugate selected from the group consisting of clotting agents, growthfactors, and antibiotics. In another embodiment, the clotting agents isthe group selected from the group consisting of thrombin and fibrin.

In another aspect, the invention provides a corneal shield containing acollagen mimetic peptide conjugate and a polymer. In one embodiment, theCMP conjugate comprises an antibiotic, an anti-inflammatory, or a smallmolecule.

In another aspect, the invention provides a wound healing devicecontaining a collagen mimetic peptide conjugate and a polymer. In oneembodiment, the CMP conjugate comprises an antibiotic, a cell adhesionmolecule, a growth factor, a component of the extracellular matrix, ananti-inflammatory, and a small molecule. In another embodiment, thedevice is in the form of a plug, mesh, strip, suture, dressing, patch,threads, suture, or biological fastener. In other embodiments, thedevice further contains a cell selected from the group consisting ofendothelial cells, dendritic cells, stem cells or other multipotentprogenitor cells, skin cells, liver cells, heart cells, kidney cells,pancreatic cells, lung cells, bladder cells, stomach cells, intestinalcells, cells of the urogenital tract, breast cells, skeletal musclecells, skin cells, bone cells, cartilage cells, keratinocytes,hepatocytes, gastro-intestinal cells, epithelial cells, endothelialcells, mammary cells, skeletal muscle cells, smooth muscle cells,parenchymal cells, osteoclasts, and chondrocytes.

In another aspect, the invention provides a kit containing the woundhealing device of any previous aspect.

In another aspect, the invention provides matrix containing a polymer inassociation with a collagen mimetic peptide, where the matrix promotesthe survival, differentiation, or proliferation of a cell. In oneembodiment, the polymer is in a hydrogel, is biochemically inert, or isphotopolymerizable. In another embodiment, the polymer is collagen,poly(ethylene oxide) diacrylate (PEODA), poly(ethylene glycol),polyvinyl alcohol, or polyacrylic acid. In one embodiment, the physicalinteraction occurs by helical assembly or strand invasion. In anotherembodiment, collagen mimetic peptide conjugate (e.g., a conjugatecontaining a reactive group, such as an acrylate group (e.g., PEG mono-or diacrylate)) acts as a cross-linking agent. In another embodiment,the matrix is susceptible to proteolytic breakdown. In anotherembodiment, the matrix further comprises an antibiotic, a cell adhesionmolecule, a contrast agent, a detectable label, a growth factor, acomponent of the extracellular matrix, an anti-inflammatory, a polymer,PEG, and a small molecule. In another embodiment, CMP is conjugated toan antibiotic, a cell adhesion molecule, a growth factor, a component ofthe extracellular matrix, an anti-inflammatory, a polymer, PEG, and asmall molecule.

In other embodiment of the above aspects, PEG is star shaped PEG, amulti-armed PEG, a graft linear PEG, PEG₂₀₀₀, or PEG₅₀₀₀. In still otherembodiments of the above aspects, the matrix further contains a cellselected from the group consisting of endothelial cells, dendriticcells, stem cells or other multipotent progenitor cells, skin cells,liver cells, heart cells, kidney cells, pancreatic cells, lung cells,bladder cells, stomach cells, intestinal cells, cells of the urogenitaltract, breast cells, skeletal muscle cells, skin cells, bone cells,cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells,epithelial cells, endothelial cells, mammary cells, skeletal musclecells, smooth muscle cells, parenchymal cells, osteoclasts, andchondrocytes.

In another aspect, the invention provides a method for synthesizing amatrix, the method involving contacting a collagen mimetic peptide to apolymer and cross-linking the polymer such that a molecular matrix isformed. In one embodiment, the polymer is in a hydrogel, isbiochemically inert, or is photopolymerizable. In another embodiment,the polymer is selected from the group consisting of poly(ethyleneglycol), poly(ethylene oxide) diacrylate (PEODA), polyvinyl alcohol, orpolyacrylic acid.

In another aspect, the invention provides a method for promoting cellsurvival or proliferation, involving growing a cell in contact with amatrix containing a polymer and a collagen mimetic peptide. In oneembodiment, the collagen mimetic peptide is capable of retaining acell-secreted collagen. In another embodiment, the matrix furthercomprises an antibiotic, a cell adhesion molecule, a contrast agent, adetectable label, a growth factor, a component of the extracellularmatrix, an anti-inflammatory, a polymer, PEG, and a small molecule. Inanother embodiment, the CMP is conjugated to an antibiotic, a celladhesion molecule, a growth factor, a component of the extracellularmatrix, an anti-inflammatory, a polymer, PEG, and a small molecule. Inanother embodiment, the polymer is in a hydrogel, is biochemicallyinert, or is photopolymerizable. In another embodiment, the cell isselected from the group consisting of endothelial cells, dendriticcells, stem cells or other multipotent progenitor cells, skin cells,liver cells, heart cells, kidney cells, pancreatic cells, lung cells,bladder cells, stomach cells, intestinal cells, cells of the urogenitaltract, breast cells, skeletal muscle cells, skin cells, bone cells,cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells,epithelial cells, endothelial cells, mammary cells, skeletal musclecells, smooth muscle cells, parenchymal cells, osteoclasts, andchondrocytes.

In yet another aspect, the invention provides a method for replacing adamaged or absent tissue, the method involving growing a cell in amatrix of any one of a previous aspect; and contacting a damaged tissueor site of an absent tissue with the cell and matrix, such that the celland matrix replaces the damaged or absent tissue.

In yet another aspect, the invention provides a method for or for tissueaugmentation, the method involving growing a cell in a matrix of aprevious aspect; and contacting a tissue that requires augmentation withthe cell and matrix, such that the cell and matrix augments the tissue.

In various embodiments of the above aspects, the cell is derived from orthe tissue is selected from any one or more of muscle, cartilage, heart,bladder, brain, nervous tissue, glial tissue, esophagus, fallopian tube,heart, pancreas, intestines, gallbladder, kidney, liver, lung, ovaries,prostate, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, breast, skeletalmuscle, skin, bone, and cartilage. In still other embodiments of theabove aspects, the cell is any one or more of endothelial cells,dendritic cells, stem cells, multipotent progenitor cells, skin cells,liver cells, heart cells, kidney cells, pancreatic cells, lung cells,bladder cells, stomach cells, intestinal cells, cells of the urogenitaltract, breast cells, skeletal muscle cells, skin cells, bone cells,cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells,epithelial cells, endothelial cells, mammary cells, skeletal musclecells, smooth muscle cells, parenchymal cells, adipocytes, osteoclasts,and chondrocytes. In another embodiment, the method is useful forcosmetic surgery. In another embodiment, the method reconstructs abreast, a face, or a body part after cancer surgery or trauma.

In yet another aspect, the invention provides a method for promotingcartilage repair, the method involving growing a chondrocyte in a matrixof a previous aspect; and contacting cartilage with the chondrocyte andmatrix, where the contact promotes cartilage repair.

In various embodiments of any of the above aspects, the collagen mimeticpeptide or collagen mimetic peptide conjugate comprises aZ-[X-Y-Gly]_(n) repeat unit (SEQ ID NO: 1), where Z is any amino acid, Xis proline or modified praline (e.g., 4-hydroxyl proline, 4-fluoropraline), Y is proline or modified proline (e.g., 4-hydroxyl proline,4-fluoro praline), and n is an integer between 1 and 20. In otherembodiments of any of the above aspects, the collagen mimetic peptidecomprises (ProHypGly)_(x) (SEQ ID NO: 10), (ProProGly)_(x) (SEQ ID NO:11), or (ProFlpGly)_(x) (SEQ ID NO: 12), where Hyp is hydroxyl proline,Flp is 4-fluoro proline, and x is any integer between 1 and 30 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30). In yetother embodiments of any of the above aspects, the collagen mimeticpeptide comprises at least 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.In still other embodiments of any of the above aspects, the collagenmimetic peptide is conjugated to an antibiotic (e.g., penicillin,tetracycline, plectasin, LAH4), a cell adhesion molecule (e.g.,cadherin, fibronectin, integrin, laminin, selectin), a contrast agent(e.g., a gadolinium complex, gadodiamide derivative, ferric ammoniumcitrate, and mangafodipar trisodium), a detectable label (e.g., acolloidal particle, an enzyme, an electron-dense reagent, a fluorescentdye, a hapten, an immunogen, a magnetic bead, a radiolabel,carboxy-fluorescein), a growth factor that promotes angiogenesis, cellgrowth, differentiation, proliferation, neurogenesis, osteogenesis, stemcell renewal, or cell survival, such as angiogenin, erythropoietin,vascular endothelial growth factor (VEGF), granulocyte/macrophage colonystimulating factor, macrophage-colony stimulating factor,platelet-derived endothelial cell growth factor, or platelet-derivedgrowth factor, a component of the extracellular matrix (e.g., collagen,elastin, fibrillin, fibronectin, laminin; proteoglycans, hyaluronan,chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratansulfate, and aggrecan), an anti-inflammatory (e.g., corticosteroids,NSAIDS), a polymer (e.g., collagen, poly(ethylene oxide) diacrylate(PEODA), poly(ethylene glycol) (PEG) (e.g., a star shaped PEG, amulti-armed PEG, a graft linear PEG, PEG₂₀₀₀, and PEG₅₀₀₀), and a smallmolecule, such as an anti-thrombotics (e.g., heparin-CMP, Hirudin-CMP,Saratin-CMP), atherosclerosis therapeutic (e.g., cholestyramine,colestipol, nicotinic acid, gemfibrozil, probucol, atorvastatin,lovastatin), a cartilage repair agent (e.g., chondroitan sulfate). Invarious embodiments, the collagen mimetic peptide binds to any one ormore collagen selected from the group consisting of type 1-29 collagen,such as type I, II, III, IV, IX, X, or XI. In yet other embodiments ofthe above aspects, collagen mimetic peptide peptide comprises an aminoacid sequence selected from the group consisting of Gly₃-(ProHypGly)₆(SEQ ID NO: 13), Gly₃-(ProHypGly)₇ (SEQ ID NO: 14), Gly₃-(ProHypGly)₈(SEQ ID NO: 15), Gly₃-(ProHypGly)₉ (SEQ ID NO: 16), (ProHypGly)₆-Tyr(SEQ ID NO: 17) (ProHypGly)-7-Tyr (SEQ ID NO: 18), (ProHypGly)-8-Tyr(SEQ ID NO: 19), Cys-(Pro-Hyp-Gly)₃ (SEQ ID NO: 20), Cys-(Pro-Hyp-Gly)₅(SEQ ID NO: 21), and Cys-(Pro-Hyp-Gly)₇ (SEQ ID NO: 22),_(carboxyfluorescein)-Gly₃-(Pro-Hyp-Gly)₆ (SEQ ID NO: 23)carboxyfluorescein-Gly₃-lys-(Pro-Hyp-Gly)₈ (SEQ ID NO: 24)PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 25), MethoxyPEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6),(Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO: 26),[AcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈]₄-star shaped PEG (core sequencedisclosed as SEQ ID NO: 27), [CF-Gly₃-Lys-Gly₃-(Pro-Hyp-Gly)_(s)]₄-starshaped PEG (core sequence disclosed as SEQ ID NO: 28),AcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 29),FL-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 30), and 5 carboxyfluorescein-Gly₃-(Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO: 31). In variousembodiments of the above aspects, the peptide has a melting transitiontemperature between 5° C. and 95° C. (e.g., between 10° C. and 80° C.,15° C. and 40° C., 20° C. and 37° C.). In various embodiments of theabove aspects, CMP is conjugated to a multi-armed PEG compound. In otherembodiment, PEG comprises an amino group, a thiol group, ismonoacrylated, or is diacrylated. In yet other embodiments of any of theabove aspects, CMP is conjugated to a clotting agent, such as thrombinor fibrin. In yet other embodiments of the above aspects, the polymer iscollagen, poly(ethylene oxide) diacrylate (PEODA), poly(ethyleneglycol), polyvinyl alcohol, or polyacrylic acid. In still otherembodiments of the above aspects, the CMP physically interacts or bindscollagen. In other embodiments, the physical interaction occurs byhelical assembly or strand invasion. In still other embodiments of theabove aspects the collagen mimetic peptide conjugate (e.g., a conjugatecontaining a reactive group, such as an acrylate group (e.g., PEG mono-or diacrylate)) acts as a cross-linking agent. In still otherembodiments of any of the above aspects, the polymer is in a hydrogel,is biochemically inert, or is photopolymerizable

By “collagen” is meant a protein component of an extracellular matrixhaving a tertiary structure that includes polypeptide chainsintertwining to form a collagen triple helix or having a characteristicamino acid composition comprising Gly-X-Y repeat units, or a fragmentthereof. Collagens useful in the methods of the invention include anycollagen known in the art (e.g., one of collagen type 1-29).

By “collagen mimetic peptide” (CMP) is meant a peptide that is able toform a collagen triple helical structure and physically interacts with acollagen polypeptide. In general a CMP has an amino acid sequencecomprising -[X-Y-Gly]_(n) repeat units. In general a CMP binds collagenwith high affinity.

A “collagen mimetic peptide conjugate” is a CMP covalently bound toanother molecule. Molecules capable of acting as CMP conjugates include,but are not limited to, polypeptides, or fragments thereof, nucleic acidmolecules, small molecule compounds, detectable labels, nanoparticles,and polymers.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in theexpression levels of a gene or polypeptide as detected by standard artknown methods such as those described above. As used herein, analteration includes a 10% change in expression levels, preferably a 25%change, more preferably a 40% change, and most preferably a 50% orgreater change in expression levels.”

“Biological sample” as used herein refers to a sample obtained from abiological subject, including sample of biological tissue or fluidorigin, obtained, reached, or collected in vivo or in situ, thatcontains or is suspected of containing nucleic acids or polypeptides.Such samples can be, but are not limited to, organs, tissues, fractionsand cells isolated from mammals including, humans such as a patient,mice, and rats. Biological samples also may include sections of thebiological sample including tissues, for example, frozen sections takenfor histologic purposes.

By “detectable label” is meant a composition that when linked to amolecule of interest renders the latter detectable, via spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive isotopes, magnetic beads,metallic beads, colloidal particles, fluorescent dyes, electron-densereagents, enzymes (for example, as commonly used in an ELISA), biotin,digoxigenin, or haptens.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.

By “an effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative in an untreated patient. The effectiveamount of active compound(s) used to practice the present invention fortherapeutic treatment of a neurodegenerative disease varies dependingupon the manner of administration, the age, body weight, and generalhealth of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “immunological assay” is meant an assay that relies on animmunological reaction, for example, antibody binding to an antigen.Examples of immunological assays include ELISAs, Western blots,immunoprecipitations, and other assays known to the skilled artisan.

By “polymer” is meant a natural or synthetic organic molecule formed bycombining smaller molecules in a regular pattern.

By “small molecule” is meant any chemical compound.

By “modulation” is meant any alteration (e.g., increase or decrease) ina biological function or activity.

By “nanoparticle” is meant an aggregate of anywhere from a few hundredto tens of thousands of atoms that have a diameter ranging from 3-300nanometers.

By “matrix” is meant a substance that fills the spaces between isolatedcells in culture. For some applications, a matrix is an adhesivesubstrate used to coat a glass or plastic surface prior to cell culture.

By “repelling cell adhesion” is meant decreasing an adhesivecharacteristic of a composition relative to an untreated composition.

By “reference” is meant a standard or control condition.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “physical interaction” is meant an association that does not requirecovalent bonding. In one embodiment, a physical interaction includesincorporation into a helical structure.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The invention provides modified collagen and related therapeuticmethods. Other features and advantages of the invention will be apparentfrom the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows circular dichroism (CD) thermal melting curves of collagenmimetic peptide derivatives 1˜3 (SEQ ID NOS 37, 36 and 38, respectively,in order of appearance). The values of Tm (° C.) are determined from thetemperature at the midpoint of the thermal transition curves.

FIG. 2 shows a CD thermal melting curve of CF-Gly₃-(PHG)₆ (peptide 4)(SEQ ID NO: 23).

FIG. 3 shows a CD thermal melting curve of mPEG₂₀₀₀-Gly₃-(PHG)₇ (peptide5) (SEQ ID NO: 6).

FIG. 4 shows a CD measurement of collagen films after the treatment withpeptide 2 or blank solution. Both solutions were pre-equilibrated at 80°C. before addition to the collagen film. Treatment with blank solution(80° C.) unfolded the native collagen and only 32% of collagen'soriginal helical content remains after the treatment. However collagenfilm treated with peptide 2 retains 80% of its original helical content.The additional helical content is likely due to peptide 2 associatingwith partially unfolded collagen in the form of triple helix.

FIGS. 5A and 5B are graphs showing the fluorescence intensities ofcollagen films (type I, bovine) treated with 5CF labeled CMPs and othercontrol samples. The X axis represents the temperature at whichfluorescence solutions were equilibrated prior to addition to thecollagen film (see support information). FIG. 5A shows the binding ofpeptide 2 and control samples to collagen (groups a and b) and gelatin(group c) films (SEQ ID NOS 37-38, respectively, in order ofappearance). The gelatin film was prepared by subjecting the collagenfilm to heat (80° C.) for 30 minutes. FIG. 5B shows the binding ofpeptide 4 (SEQ ID NO: 39) to collagen films.

FIG. 6 shows transmission electron and fluorescence (inset) micrographsof collagen fibers (type I) after the treatment with peptide 4 at 30° C.The collagen fibers exhibit native banding pattern suggesting that themodification process did not disrupt the native collagen structure. Thepresence of peptide 4 on the collagen fiber was confirmed byfluorescence microscopy (inset).

FIGS. 7A, 7B and 7C are optical micrographs of human fibroblasts (FIGS.7A and 7B) and breast epithelial cells (FIG. 7C) cultured on collagenfilms that were pre-treated with mPEG₂₀₀₀ (FIG. 7A), or peptide 5 (FIGS.7B and 7C). Areas of the picture to the right side of the dotted lineswere treated with mPEG₂₀₀₀ or peptide 5.

FIG. 8 is a schematic diagram showing methods for altering the growth ofepithelial cells on a collagen gel using modified CMP conjugated to PEG.

FIGS. 9A-9C are optical micrographs of human breast epithelial cellscultured on collagen films that were treated withmPEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly), (SEQ ID NO: 6) 3rd day (FIG. 9A), 5th day(FIG. 9B), and 7th day (FIG. 9C). The bar scales represent 100 μm.

FIGS. 10A-10F are optical micrographs of human breast epithelial cellscultured on collagen films that were treated with(Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO: 26) 3rd day (A), 5th day (B),and 7th day (C), or longer 10D-10F. The bar scales represent 250 μm.

FIG. 11 is a graph that quantitates the release of fluorescence-labeledCMP derivatives from collagen films incubated in PBS buffer solution ofpH 7.4 at 37° C. FIG. 11 discloses SEQ ID NOS 46-49 and 46-49,respectively, in order of appearance.

FIG. 12 is a schematic diagram showing the structure of star shaped,rigid and flexible linear PEG on substrate. The structure of star shapedor suitable linear PEG (PEG5000) explains its capacity for repellingapproaching molecules or cells compared to flexible linear PEG(PEG20000).

FIG. 13 is a schematic diagram showing the structures of CMP-star shapedPEG. A) [AcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈]₄-star shaped PEG (coresequence disclosed as SEQ ID NO: 27). B)[CF-Gly₃-Lys-Gly₃-(Pro-Hyp-Gly)₈]₄-star shaped PEG wore sequencedisclosed as SEQ ID NO: 28), fluorescence tagged star shaped PEG-CMP.Star shaped PEG: NHS activated four-armed star shaped PEG, molecularweight is approximately 10,000 Da; CMP:AcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 29); CF-CMP:carboxyfluorescein-Gly₃-lys-(Pro-Hyp-Gly)₈ (SEQ ID NO: 24).

FIG. 14 shows the thermal melting transition curves for peptides 1′˜4′.

FIG. 15 shows the Gaussian fit of the dynamic light scattering (DLS)data showing the hydrodynamic size of nanoparticle-peptide number(NP-Xs).

FIG. 16 shows the determination of the number of peptides per Au NP.

FIGS. 17A-17D are transmission electron microscope (TEM) micrographs ofnegatively stained NP-1 (FIG. 17A), NP-2 (FIG. 17B), NP-3 (FIG. 17C) andunmodified NPs (FIG. 17D). Scale bar=50 nm

FIGS. 18A and 18B show the aggregation parameters of NP-3 at differentNaCl concentration (FIG. 18A) and pH (FIG. 18B).

FIGS. 19A-19C show TEM micrographs of reconstituted type I collagenfibers after incubation with NP-4 (FIG. 19A) and NP-3 (FIG. 19B) at 25°C. NP-3 shows preferential affinity to the dark bands of collagen fiber(FIG. 19C). Scale bar=500 nm.

FIG. 20 shows a TEM micrograph of NP-3 incubated with reconstituted typeI collagen fibers at 40° C.

FIG. 21 is a schematic diagram showing the general approach used forcollagen mimetic peptide (CMP) functionalized gold nanoparticle (NPs)use. CMP functionalized NPs are highly stable in aqueous solution andexhibit preferential affinity to the gap regions of intact type Icollagen fibers. CMP functionalized NPs are used to identify structuralabnormalities in collagen fibers that are related to many debilitatinghuman diseases. FIG. 21 discloses SEQ ID NO: 50.

FIG. 22 is a schematic diagram showing a CMP/poly(ethylene oxide)diacrylate (PEODA) hydrogel containing cells. The collagen secreted fromthe cell binds efficiently to the CMP present in the hydrogel matrix.

FIGS. 23A and 23B show the effects of CMP on collagen retention. FIG.23A is a schematic diagram showing that cell-secreted collagen is moreeffectively retained by hydrogels that include CMP (right panel) than byhydrogels lacking CMP (left panel). FIG. 23B is a graph showinghydroxyproline content measured (n=3) in a hydrogel following one weekof cell culture. Controls were as PEODA hydrogel alone without modifiedPEG-CMP.

FIG. 24 is a fluorescence micrograph of chondrocytes encapsulated in 2%CMP/PEODA hydrogels after Live/Dead staining.

FIGS. 25A-25F are micrographs of chondrocytes cultured in PEODA, 1%CMP/PEODA, and 2% CMP/PEODA matrices. Histological sections wereevaluated after 2 weeks of culture. Safranine-O staining forglycosaminoglycan (FIG. 25A, 25B, and 25C) and Masson Trichrome stainingfor collagen (FIG. 25D, 26E, and 26F) were used.

FIGS. 26A-26C are micrographs showing immunohistochemical staining ofchondrocytes cultured in matrices containing PEODA control, 1%CMP/PEODA, and 2% CMP/PEODA. Antibody for collagen type H was used.Controls showed no staining for antigen.

FIGS. 27A and 27B are graphs showing the quantitation of extracellularmatrix secretions of chondrocytes as evaluated by biochemical assays(n=3, *: p<0.05, **: p<0.0005, ***: p<0.0001): for glycosaminoglycancontent (FIG. 27A); or total collagen content (FIG. 27B). Backgroundlevels of collagen content present in the acellular CMP hydrogels wassubtracted from these totals.

FIG. 28 provides a schematic diagram showing monoacrylated PEG-CMP.Monoacrylated PEG-CMP is polymerized with difunctional PEG to form ahydrogel.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions comprising modified collagen andrelated therapeutic and diagnostic methods. In contrast with previousmethods, which typically rely on chemical modifications, particularlycovalent modifications, the methods described herein provide for thephysical modification of collagen. This invention is based, in part, onthe discovery that collagen mimetic peptides (CMPs) of sequence-(Pro-Hyp-Gly)_(x)- exhibited strong affinity for collagen. In addition,the invention provides for the modulation of the cell adhesioncharacteristics of collagen by contacting a poly(ethyleneglycol)-CMPconjugate with collagen.

Collagen

Collagen is the most abundant structural protein in the body, existingas the foremost component of the extracellular matrix (ECM). Most typesof collagen contain a unique tertiary structure that includes threeindividual right-handed helical polypeptide chains intertwining to forma left-handed helix. Collagen has a characteristic amino acidcomposition comprised of Gly-X-Y repeat units, where 28% of the Xpositions consist of proline (Pro) and 38% of the Y positions consist ofpost-translationally modified 4-hydroxyproline residues (Hyp). Thetriple-helical structure can be denatured upon heating above its meltingtemperature¹. Collagen mimetic peptides (CMPs) with Gly-X-Y sequenceshave been used to analyze collagen. CMPs based on Pro-Pro-Gly andPro-Hyp-Gly trimers have been widely studied, and their collagen-liketriple helical structure and melting behaviors have beencharaterized²⁻⁸. Unlike collagen, CMPs exhibit reversible meltingbehavior due to their small size. When denatured collagen is cooled, itregains only about 5-10% of its original triple helical content, and theremainder turns into gelatin. In contrast, CMP regains almost 100% ofits original triple helical structure after denaturation.

Collagen is used in a variety of medical applications includinghemostatic materials, biocompatible coatings, drug delivery and tissueengineering. Collagen-based biomaterials are also used in soft-tissueengineering and repair. In the past two decades, a multitude of medicalproducts composed of collagen have been approved by the FDA, and manyare available as commercial products⁹, including collagen-based cornealshields, anti-infectious catheters, tissue sealants, hemostatic sponges,and topical wound dressing products. Collagen is also used as a tissueengineering substrate for skin¹⁰, bone¹¹, and blood vesselreplacement¹². Collagen has typically been used as a scaffold to supporttissue growth and promote healing or for the development of engineeredtissues in organ replacement therapies. In virtually any application,wherever collagen is used, CMP modified collogen maybe substituted.

Collagen and Human Disease

Collagens are complex molecules that provide structure, strength, andelasticity (the ability to stretch) to connective tissue. Mutations inI, II, III, IX, X, and XI collagens are associated with a variety ofhuman connective tissue disorders affecting bone, cartilage, and bloodvessels. Ehlers-Danlos syndrome (EDS) is most often associated with agenetic defect in type I collagen that is characterized by skinhyperextensibility, fragile and soft skin, delayed wound healing withformation of atrophic scars, easy bruising, and generalized jointhypermobility. Osteogenesis imperfecta or “brittle bone disease” arisesfrom mutations in two genes encoding type I collagen. People withosteogenesis imperfecta (OI) have bones that fracture easily, low musclemass, and joint and ligament laxity. There are four major types of OIranging in severity from mild to lethal. The appearance of people withOI varies considerably. Individuals may also have a blue or gray tint tothe sclera (whites of the eyes), thin skin, growth deficiencies, andfragile teeth. They may develop scoliosis, respiratory problems, andhearing loss.

Type II and type XI collagens are components of the cartilage found injoints and the spinal column, the inner ear, and the vitreous humor ofthe eye. The type H and XI collagenopathies include achondrogenesis type2, hypochondrogenesis, Kniest dysplasia, otospondylomegaepiphysealdysplasia, spondyloepimetaphyseal dysplasia, Strudwick type,spondyloepiphyseal dysplasia congenita spondyloperipheral dysplasia,Stickler syndrome, and Weissenbacher-Zweymüller. The clinical featuresof the type II and XI collagenopathies typically include problems withbone development that results in short stature, enlarged joints, spinalcurvature, and juvenile arthritis. Problems with vision and hearing, aswell as a cleft palate with a small lower jaw, are common.

Diagnostics

Given the propensity of collagen mimetic peptides to interact withcollagen, compositions of the invention can be used to visualize thestructure of collagen by binding a collagen mimetic peptide conjugate tocollagen present in a patient sample. In one embodiment, compositions ofthe invention are used for the diagnosis of a disease or disordercharacterized by an alteration in the structure of collagen. In oneapproach, a nanoparticle having a collagen mimetic peptide, or collagenmimetic peptide conjugate is used as a diagnostic. Such nanoparticlesare used to examine the structure of collagen in biological samplesderived from patients suspected of having a connective tissue disorderrelated to the disruption of collagen. The collagen mimetic peptideallows for the physical association of the nanoparticle with a collagen(e.g., type 1-29) present in the tissue sample. The tissue sample isthen analysed using a transmission electron microscopy. Undertransmission electron microscopy, collagen fibers exhibit characteristicbanding patterns. CMP-NPS bind to defined locations within the collagenfiber. This binding pattern provides an indication of the structuralintegrity of the collagen molecules and their assembly. Alterations inthe structural integrity can be visualized with CMP-NP and correlatedwith particular connective tissue disease states. Thus, CMP-NP areuseful as diagnostics for the identification of alterations in collagentypes 1-29.

In other embodiments, compositions of the invention are used to identifythe presence of a thrombosis comprising collagen in a vessel or toidentify vessels having an increased risk of having a thrombosis.Vessels that have been subjected to angioplasty are particularly proneto the formation of a thrombosis.

Collagen Mimetic Peptide Conjugates

Collagen mimetic peptides may be conjugated to a variety of agents usingmethods known in the art and described herein. Typically, thisconjugation is mediated by a covalent bond. Suitable agents includetherapeutic and diagnostic agents. Such agents include, but are notlimited to, antibiotics, anti-thrombotics, cell adhesion molecules,components of the extracellular matrix, contrast reagents, detectablelabels, growth factors, polymers, PEG, and small compounds havingbiological activity.

Antimicrobial Agents

Any antimicrobial agent known in the art can be used in the compositionsof the invention at concentrations generally used for such agents.Antimicrobial agents include antibacterials, antifungals, andantivirals.

Exemplary antibiotics (i.e., antibacterial agents) include thepenicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, andamoxicillin), the cephalosporins (e.g., cefadroxil, ceforanid,cefotaxime, and ceftriaxone), the tetracyclines (e.g., doxycycline,minocycline, and tetracycline), the aminoglycosides (e.g., amikacin,gentamycin, kanamycin, neomycin, streptomycin, and tobramycin), themacrolides (e.g., azithromycin, clarithromycin, and erythromycin), thefluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin),and other antibiotics including chloramphenicol, clindamycin,cycloserine, isoniazid, rifampin, and vancomycin.

In particular embodiments, a penicillin-CMP antibiotic has the followingstructure:

In another embodiment, a tetracycline-CMP antibiotic has the followingstructure:

In still other embodiments, the antimicrobial is a plectasin-CMP or anLAH4-CMP having the following sequences, respectively:

Plectasin-CMP (antimicrobial) (SEQ ID NO: 32)GFGCNGPWDEDDMQCHNHCKSIKGYKGGYAKGGFVCKCY-CMP LAH4-CMP (SEQ ID NO: 33)KKALLALALHHLAHLALHLALALKKA-CMP.

Antiviral agents are substances capable of inhibiting the replication ofviruses. Examples of anti-viral agents include1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9-2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon, adenine arabinoside, proteaseinhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesisinhibitors, structural protein synthesis inhibitors, attachment andadsorption inhibitors, and nucleoside analogues such as acyclovir,penciclovir, valacyclovir, and ganciclovir.

Antifungal agents include both fungicidal and fungistatic agents suchas, for example, benzoic acid, undecylenic alkanolamide, ciclopiroxolamine, polyenes, imidazoles, allylamine, thicarbamates, amphotericinB, butylparaben, clindamycin, econaxole, fluconazole, flucytosine,griseofulvin, nystatin, and ketoconazole.

Atherosclerosis Therapeutics

In other embodiments, a CMP conjugate is used for the treatment ofatherosclerosis. For such applications a CMP is conjugated, for exampleto an anti-platelet medication (e.g., aspirin), anti-coagulants (e.g.,as heparin, warfarin, aspirin, ticlopidine, and clopidogrel) orinhibitors of platelet clumping. Other atherosclerosis therapeuticsinclude, but are not limited to, cholestyramine, colestipol, nicotinicacid, gemfibrozil, probucol, atorvastatin, lovastatin.

Anti-Thrombotics

The collagen mimetic peptides are also useful for the delivery ofcompounds having anti-thrombotic activity. Such compounds includeheparin, hirudin, ReoPro™, Streptokinase, urokinase, and tissueplasminogen activator (t-PA). Other drugs include recombinant, orgenetically engineered, t-PA (a newer version of t-PA) and TNK(Tenecteplase) or other anti-thrombotic compounds. In one particularembodiment, the anti-thrombotic is PEG-CMP having the followingstructure:

In still other embodiments, the anti-thrombotic is a saratin-CMP or ahirudin CMP conjugate having the following sequences, respectively:

Saratin-CMP (anti-platelet) (SEQ ID NO: 34)EEREDCWTFYANRKYTDFDKSFKKSSDLDECKKTCFKTEYCYIVFEDTVNKECYYNVVDGEELDQEKFVVDENFTENYLTDCEGKDAGNAAGTGDESDEV DED-K-(CMP) ₂Hirudin-CMP (anti-platelet) (SEQ ID NO: 35)LTYTDC(6)TESGQNLC(14)LC(16)EGSNVC(22)GQGNKC(28)ILGSDGEKNQC(39)VTGEGTPKPQSHNDGDFEEIPEEY(SO3)LQ-K- (CMP) ₂.

Cell Adhesion Molecules

Cell adhesion molecules suitable for CMP conjugation include componentsof the extracellular matrix that promote cell spreading or extension orfragments thereof. Preferably, fragments of a cell adhesion moleculeinclude the adhesion molecule binding domain. Such binding domainsinclude consensus sequences that mediate cell-cell interactions,including the RGD peptide. Integrins bind the RGD motif in cellattachment proteins (See, for example, Tosatti et al., J Biomed MaterRes. 68(3):458-72, 2004; and Reyes et al., J Biomed Mater Res69:591-600, 2004, which are hereby incorporated by reference). Suchmolecules may be conjugated to a collagen mimetic peptide of theinvention for use in the therapeutic compositions described herein.Exemplary cell adhesion molecules include cadherin (e.g., E-cadherin,N-cadherin), cell adhesion molecule (CAM) (e.g., vascular cell adhesionmolecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 neuronalcell adhesion molecule), fibronectin, integrin (e.g., B-integrin),laminin, and selectin.

Growth Factors

Growth factors are typically polypeptides or fragments thereof thatsupport the survival, growth, or differentiation of a cell. A collagenmimetic peptide described herein can be conjugated to virtually anygrowth factor known in the art. Such growth factors includeangiopoietin, acidic fibroblast growth factors (aFGF) (GenBank AccessionNo. NP_(—)149127) and basic FGF (GenBank Accession No. AAA52448), bonemorphogenic protein (GenBank Accession No. BAD92827), vascularendothelial growth factor (VEGF) (GenBank Accession No. AAA35789 orNP_(—)001020539), epidermal growth factor (EGF) (GenBank Accession No.NP_(—)001954), transforming growth factor α (TGF-α) (GenBank AccessionNo. NP_(—)003227) and transforming growth factor β (TFG-β) (GenBankAccession No. 1109243A), platelet-derived endothelial cell growth factor(PD-ECGF) (GenBank Accession No. NP_(—)001944), platelet-derived growthfactor (PDGF) (GenBank Accession No. 1109245A), tumor necrosis factor α(TNF-α) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF)(GenBank Accession No. BAA14348), insulin like growth factor (IGF)(GenBank Accession No. P08833), erythropoietin (GenBank Accession No.P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF)(GenBank Accession No. AAB59527), granulocyte/macrophage CSF (GM-CSF)(GenBank Accession No. NP_(—)000749) and nitric oxide synthase (NOS)(GenBank Accession No. AAA36365).

Components of the Extracellular Matrix

In still other embodiments, a CMP is conjugated to a component of theextracellular matrix (ECM). ECM components include structural proteins,such as collagen and elastin; proteins having specialized functions,such as fibrillin, fibronectin, and laminin; and proteoglycans thatinclude long chains of repeating disaccharide units termed ofglycosaminoglycans (e.g., hyaluronan, chondroitin sulfate, dermatansulfate, heparan sulfate, heparin, keratan sulfate, aggrecan).

Anti-Inflammatories

In other embodiments, a CMP is conjugated to an anti-inflammatory agent.Such anti-inflammatory agents include, but are not limited to,Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; AlphaAmylase; Amcinafal; Amcinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; and Zomepirac Sodium.

In other embodiments, a CMP is conjugated to an agent having diagnosticapplications. Such agents include contrast reagents and detectablelabels.

Contrast Reagents

Magnetic resonance imaging (MRI) is typically used for diagnosticpurposes to visualized diseased organs or tissues. The utility of MRI ishampered by poor image quality. Collagen mimetic peptides can beconjugated to conventional MRI contrast reagents and then introduced toa desired tissue or tissue sample where the collagen-contrast reagentconjugate binds to collagens present in the tissue to enhance MRI imagequality. Exemplary contrast reagents include gadolinium complex,gadodiamide derivative, ferric ammonium citrate, and mangafodipartrisodium.

Detectable Labels

Detectable labels include compositions that when linked to a collagenmimetic peptide render the peptide detectable via spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Exemplarydetectable labels include radioactive isotopes, magnetic beads, metallicbeads, colloidal particles, fluorescent dyes, electron-dense reagents,enzymes (for example, as commonly used in an ELISA), biotin,digoxigenin, and haptens.

Polymers

Virtually any polymer known in the art may be conjugated to a CMP. Inone embodiment, attachment occurs via an acrylate group (e.g., PEGmonoacrylate, PEG diacrylate, see FIG. 28). Polymers that arebiocompatible, non-immunogenic, or that support cell survival orproliferation are preferred. Natural or synthetic polymers capable offorming a matrix are particularly useful. A matrix is a substance thatfills the spaces between isolated cells in culture. For someapplications, a matrix is an adhesive substrate used to coat a glass orplastic surface prior to cell culture. For other applications, cells areembedded in a matrix, or injected into a matrix already implanted at adesired site. In another approach, a matrix provides a physical supportand an adhesive substrate for isolated cells during in vitro culturingand subsequent implantation. Preferred polymers for use in a matrix havemechanical and biochemical properties that enhance the viability andproliferation of transplanted cells, tissues, or organs. The matrixconfiguration is dependent on the tissue that is to be treated,repaired, or produced, but desirably, the matrix is a pliable,biocompatible, porous template that allows for cell and/or vasculargrowth.

Preferred polymers for use in the methods of the invention includepoly(ethylene glycol) (PEG), star shaped PEG, grafted linear PEG,poly(ethylene oxide) diacrylate (PEODA), polyacrylic acid, poly vinylalcohol, collagen gels, poly (D, L-lactide-co-glycolide (PLGA) fibermatrices, polyglactin fibers, calcium alginate gels, polyglycolic acid(PGA) meshes, and other polyesters, such as poly-(L-lactic acid) (PLLA)and polyanhydrides. Matrices can include materials that arenon-biodegradable or biodegradable. Desirably, biodegradable materialswill degrade over a time period of less than a year, more preferablyless than six months.

Desirably, a collagen mimetic peptide is conjugated to a polymer capableof forming a hydrogel. A hydrogel is formed when an organic polymer(natural or synthetic) is cross-linked via covalent, ionic, or hydrogenbonds to create a three-dimensional open-lattice structure that entrapswater molecules to form a gel. Examples of materials that can be used toform a hydrogel include poly(ethylene glycol), polysaccharides (e.g.,alginate), polyphosphazenes, and polyacrylates (e.g., hydroxyethylmethacrylate). Other materials that can be used include proteins (e.g.,fibrin, collagen, fibronectin) and polymers (e.g.,polyvinylpyrrolidone), and hyaluronic acid.

In general, these polymers are at least partially soluble in aqueoussolutions, such as buffered salt solutions, or aqueous alcoholsolutions. In one embodiment, the polymer includes a charged side group,or monovalent ionic salt thereof. Examples of polymers with acidic sidegroups that can be reacted with cations are poly (phosphazenes), poly(acrylic acids), poly (methacrylic acids), copolymers of acrylic acidand methacrylic acid, poly (vinyl acetate), and sulfonated polymers(e.g., sulfonated polystyrene). Copolymers having acidic side groupsformed by reaction of acrylic or methacrylic acid and vinyl ethermonomers or polymers can also be used. Examples of acidic groups arecarboxylic acid groups, sulfonic acid groups, halogenated (preferablyfluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.Examples of polymers with basic side groups that can be reacted withanions are poly (vinyl amines), poly (vinyl pyridine), poly (vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations in water atroom temperature to form a hydrogel matrix. Additional methods for thesynthesis of the other polymers described above are known to thoseskilled in the art (see, for example, Concise Encyclopedia of PolymerScience and Polymeric Amines and Ammonium Salts, E. Goethals, editor,Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such as poly(acrylic acid), are commercially available.

In another approach, a synthetic polymer capable of forming a matrix isused. For some applications, synthetic polymers are preferred forreproducibility and controlled release kinetics. Synthetic polymers thatcan be used include biodegradable polymers, such as poly(lactide),poly(glycolic acid), poly(lactide-co-glycolide), poly (caprolactone),polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates and degradable polyurethanes,and non-erodible polymers such as polyacrylates, ethylene-vinyl acetatepolymers and other acyl substituted cellulose acetates and derivativesthereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl fluoride, poly (vinyl imidazole), chlorosulphonatedpolyolifins, polyethylene oxide, polyvinyl alcohol, Teflon and nylon.Non-degradable materials can also be used to form a matrix.

One preferred non-degradable material for implantation of a matrix is apolyvinyl alcohol sponge, or alkylation or acylation derivatives thereof(e.g., ester derivatives), including esters.

Delivery of CMP Conjugates

The invention provides a simple means for delivering biologically activecompounds (including nucleic acids, peptides, small molecule inhibitors,and mimetics) conjugated to a collagen mimetic peptide (CMP). Typically,the CMP conjugate is incorporated into a polymer matrix containingnatural or synthetic polymers. In one embodiment, the CMP conjugate isincorporated in a collagen matrix. The collagen matrix comprising theCMP conjugate is delivered to a subject and the CMP conjugate isreleased from the collagen matrix. The CMP conjugate is capable ofacting as a therapeutic for the treatment of a disease or disorder thatrequires controlled and/or localized drug delivery over some period oftime (e.g., 1, 3, 5, 7 days; 2, 3, 4 weeks; 1, 2, 3, 6, 12 months). Abiologic agent conjugated to a collagen mimetic peptide and found tohave medicinal value using the methods described herein is useful as adrug or as information for structural modification of existingcompounds, e.g., by rational drug design. Desirably, the conjugatesinclude antibiotics (e.g., penicillin, tetracycline, plectasin, LAH4),cell adhesion molecules (e.g., cadherin, fibronectin, integrin, laminin,selectin), growth factors that promote angiogenesis, cell growth,differentiation, proliferation, neurogenesis, osteogenesis, stem cellrenewal, or cell survival (e.g., angiogenin, erythropoietin, vascularendothelial growth factor (VEGF), granulocyte/macrophage colonystimulating factor, macrophage-colony stimulating factor,platelet-derived endothelial cell growth factor, and platelet-derivedgrowth factor), or small molecules, such as anti-thrombotics (e.g.,heparin-CMP, Hirudin-CMP, Saratin-CMP), anti-atherosclerosis agents,cartilage repair agents (e.g., chondroitin sulfate, glucosamine sulfate,hyaluronic acid). The polymers including the collagen mimetic peptideconjugates are administered either as liquids or solids. Where thepolymers are administered as a liquid, they are typically converted to asolid in vivo by cross-linking. Such crosslinking may be accomplishedusing any method known in the art, such as photopolymerization.

If desired, collagen mimetic peptide conjugates are incorporated intohydrogel-forming polymeric materials that are useful as drug deliverydevices. Hydrogel-forming polymers are polymers that are capable ofabsorbing a substantial amount of water to form elastic or inelasticgels. Medical devices incorporating hydrogel-forming polymers arecapable of being implanted in liquid or gelled form. Once implanted, thehydrogel forming polymer absorbs water and swells. The release of apharmacologically active agent incorporated into the device using acollagen mimetic peptide takes place through this gelled matrix via adiffusion mechanism. Many hydrogels, although biocompatible, are notbiodegradable or are not capable of being remodeled and incorporatedinto a host tissue.

For therapeutic uses, the compositions or agents identified using themethods disclosed herein may be administered systemically. In oneembodiment, a matrix comprising a CMP conjegate is formulated in apharmaceutically-acceptable buffer such as physiological saline.Preferable routes of administration include, for example, subcutaneous,intravenous, interperitoneally, intramuscular, or intradermal injectionsthat provide continuous, sustained levels of the drug in the subject.Treatment of subjects (e.g., human patients or other animals) will becarried out using a therapeutically effective amount of a CMPtherapeutic conjugate in a physiologically-acceptable carrier, such as acollagen matrix that includes the CMP therapeutic conjugate. Suitablecarriers and their formulation are described, for example, inRemington's Pharmaceutical Sciences by E. W. Martin. The amount of thetherapeutic agent, to be administered varies depending upon the mannerof administration, the age and body weight of the subject, and with theclinical symptoms of the subject. Generally, amounts will be in therange of those used for other agents used in the treatment of similardiseases (e.g., thrombosis, atherosclerosis). A compound is administeredat a dosage that controls the clinical or physiological symptoms of thedisease as determined by a diagnostic method known to one skilled in theart.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of a disease may beby any suitable means that results in a concentration of the therapeuticthat, combined with other components, is effective in ameliorating,reducing, or stabilizing the disease. The compound may be contained inany appropriate amount in a any suitable carrier substance, and isgenerally present in an amount of 1-95% by weight of the total weight ofthe composition. The composition may be provided in a dosage form thatis suitable for parenteral (e.g., subcutaneously, intravenously,intramuscularly, or intraperitoneally) administration route. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or inproximity to the tissue or organ that requires treatment; (v)formulations that allow for convenient dosing, such that doses areadministered, for example, once every one or two weeks; and (vi)formulations that target an disease by using carriers or chemicalderivatives to deliver the therapeutic agent to a particular cell typewhose function is perturbed in the disease. For some applications,controlled release formulations obviate the need for frequent dosingduring the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes that includethe CMP therapeutic conjugate.

Typically, a polymer comprising the CMP therapeutic conjugate isdelivered to the subject identified as in need of such treatment. Alarge number of polymers can be used to construct the delivery devicesof the present invention. The only requirements are that they are inert,non-immunogenic and of the desired permeability. The rate of passage ofthe drug through the material by diffusion is generally dependent on thesolubility of the drug therein, as well as on the thickness of the wall.This means that selection of appropriate materials for fabricating thewall will be dependent on the particular drug to be used. The rate ofdiffusion of the effective agent through a polymeric layer(s) of thepresent invention may be determined via diffusion studies using, forexample, a CMP therapeutic conjugate comprising a detectable label asdescribed herein.

In one embodiment, the CMP therapeutic conjugate is contained within acollagen matrix, such that the CMP therapeutic conjugate physicallyassociates with the collagen. The drug delivery devices of the inventionmay be made in a wide variety of ways, such as by obtaining an effectiveamount of a CMP therapeutic conjugate in a collagen matrix andcompressing the matrix to a desired shape. Once shaped, one or morecoating layers is applied. Such coatings are used to delay release ofthe CMP therapeutic conjugate. The drug delivery system of the inventionis administered to subject via any route of administration known in theart. Such routes of administration include intraocular, oral,subcutaneous, intramuscular, intraperitoneal, intranasal, dermal, andthe like. The drug delivery system of the invention is particularlysuitable for direct implantation.

The delivery system is disclosed for the controlled release of CMPtherapeutic conjugate from a collagen matrix into the surroundingenvironment. Controlled release delivery systems include those systemscapable of site specific delivery, extended release, sustained release,delayed release, repeat action, prolonged release, bimodal release,pulsitile release, modified delivery, pH sensitive delivery, and/ortarget specific delivery, among others.

As used herein, optionally, the system may include agents added to aidin gastric bypass or to modify the release profile due to pH-specificswelling characteristics or site-specific enzyme degradation within thegastrointestinal tract. These agents may include, but are not limitedto, at least one of alginate, polysaccharides such as such as gelatin orcollagen, guar gum, xanthan gum, pectin, heterogeneous protein mixtures,and polypeptides. The polysaccharides may be pectin and/or an alginatesalt, among others. The galactomannan gums may be guar gum, xanthan gumand/or locust bean gum, among others. The polyethylene derivatives maybe polyethylene oxide (PEO) and/or polyethylene glycol (PEG), amongothers. The hydrolyzed proteins may be gelatin and/or collagen, amongothers.

Release of the CMP conjugate into the surrounding environment may beaccomplished through a rate-controlled hydration and subsequent swellingof hydrophilic agents. The release of the CMP conjugate is determined bythe erosion rate and polymeric disentanglement of the swollenhydrophilic matrix. Typically, the swelling of the hydrophilic matrixallows for a highly reproducible, programmable release pattern. Theprogrammability of the system allows for nearly any physiologicallyrelevant release pattern to be accomplished. Formulation specific to thephysical characteristics of a CMP therapeutic conjugate and the desiredrelease profile can be accomplished through both theoretical andempirical means, allowing dissolution of the system and CMP therapeuticconjugate release to occur in a specific physiologic region. Release ofcontents in a given region of the gastrointestinal tract is accomplishedby the slowly hydrating hydrophilic matrix containing the biologicalactives segregated from the external environment until the desiredphysiologic region of release, which may be employed to achieve gastricbypass. Consideration of both the area and duration of release isessential in formulation so as to program the system with an appropriateratio of components to ensure the desired release profile.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active active inflammatorybowel disorder therapeutic(s), the composition may include suitableparenterally acceptable carriers and/or excipients. The activeinflammatory bowel disorder therapeutic(s) may be incorporated intomicrospheres, microcapsules, nanoparticles, liposomes, or the like forcontrolled release. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active CMP therapeutic conjugate(s) aredissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol or thelike.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the active drugmay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers, such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. Such formulations are known to the skilled artisan.Excipients may be, for example, inert diluents or fillers (e.g.,sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starchesincluding potato starch, calcium carbonate, sodium chloride, lactose,calcium phosphate, calcium sulfate, or sodium phosphate); granulatingand disintegrating agents (e.g., cellulose derivatives includingmicrocrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active active inflammatory boweldisease therapeutic substance). The coating may be applied on the soliddosage form in a similar manner as that described in Encyclopedia ofPharmaceutical Technology, supra.

At least two active therapeutics may be mixed together in the tablet, ormay be partitioned. In one example, the first active therapeutic iscontained on the inside of the tablet, and the second active therapeuticis on the outside, such that a substantial portion of the secondtherapeutic is released prior to the release of the first activetherapeutic.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules where the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules where the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructedto release the active therapeutic by controlling the dissolution and/orthe diffusion of the active substance. Dissolution or diffusioncontrolled release can be achieved by appropriate coating of a tablet,capsule, pellet, or granulate formulation of compounds, or byincorporating the compound into an appropriate matrix. A controlledrelease coating may include one or more of the coating substancesmentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax,carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryldistearate, glycerol palmitostearate, ethylcellulose, acrylic resins,dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride,polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3butylene glycol, ethylene glycol methacrylate, and/or polyethyleneglycols. In a controlled release matrix formulation, the matrix materialmay also include, e.g., hydrated methylcellulose, carnauba wax andstearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more therapeuticcompounds may also be in the form of a buoyant tablet or capsule (i.e.,a tablet or capsule that, upon oral administration, floats on top of thegastric content for a certain period of time). A buoyant tabletformulation of the compound(s) can be prepared by granulating a mixtureof the compound(s) with excipients and 20-75% w/w of hydrocolloids, suchas hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

CMP Conjugates Coatings

A CMP conjugate may be included in a coating material that is used tocoat a medical device (e.g., drug delivery or other medical device).Such coatings are used, for example, for altering the adehesiveproperties of the device or for drug delivery. For example, a polymericcoating, such as collagen, polyethylene glycol, polyurethane,polytetrafluoroethylene, polyalkylmethacrylates, polyarylmethacrylates,poly(ethylene-co-vinyl acetate), or any other polymer or combination ofpolymers, may be combined with a CMP of the present invention, such thatthe CMP is fixed to or physically associates with the polymer, and theCMP conjugate polymer combination is applied to a medical device. TheCMP conjugate thereby modulates the cell adhesive properties of themedical device or provides for release of a therapeutic from the device.Such coatings can be applied to any medical device known in the art,including, but not limited to, drug-delivering vascular stents (e.g., aballoon-expanded stents); other vascular devices (e.g., grafts,catheters, valves, artificial hearts, heart assist devices); implantabledefibrillators; blood oxygenator devices (e.g., tubing, membranes);surgical devices (e.g., sutures, staples, anastomosis devices, vertebraldisks, bone pins, suture anchors, hemostatic barriers, clamps, screws,plates, clips, vascular implants, tissue adhesives and sealants, tissuescaffolds); membranes; cell culture devices; chromatographic supportmaterials; biosensors; shunts for hydrocephalus; wound managementdevices; endoscopic devices; infection control devices; orthopedicdevices (e.g., for joint implants, fracture repairs); dental devices(e.g., dental implants, fracture repair devices), urological devices(e.g., penile, sphincter, urethral, bladder and renal devices, andcatheters); colostomy bag attachment devices; ophthalmic devices (e.g.intraocular coils/screws); glaucoma drain shunts; synthetic prostheses(e.g., breast); intraocular lenses; respiratory, peripheralcardiovascular, spinal, neurological, dental, ear/nose/throat (e.g., eardrainage tubes); renal devices; and dialysis (e.g., tubing, membranes,grafts), urinary catheters, intravenous catheters, small diametergrafts, vascular grafts, artificial lung catheters, atrial septal defectclosures, electro-stimulation leads for cardiac rhythm management (e.g.,pacer leads), glucose sensors (long-term and short-term), degradablecoronary stents (e.g., degradable, non-degradable, peripheral), bloodpressure and stent graft catheters, birth control devices, prostatecancer implants, bone repair/augmentation devices, breast implants,cartilage repair devices, dental implants, implanted drug infusiontubes, intravitreal drug delivery devices, nerve regeneration conduits,oncological implants, electrostimulation leads, pain managementimplants, spinal/orthopedic repair devices, wound dressings, embolicprotection filters, abdominal aortic aneurysm grafts, heart valves(e.g., mechanical, polymeric, tissue, percutaneous, carbon, sewingcuff), valve annuloplasty devices, mitral valve repair devices, vascularintervention devices, left ventricle assist devices, neuro aneurysmtreatment coils, neurological catheters, left atrial appendage filters,hemodialysis devices, catheter cuff, anastomotic closures, vascularaccess catheters, cardiac sensors, uterine bleeding patches, urologicalcatheters/stents/implants, in vitro diagnostics, aneurysm exclusiondevices, and neuropatches. Examples of other suitable devices include,but are not limited to, vena cava filters, urinary dialators, endoscopicsurgical tissue extractors, atherectomy catheters, clot extractioncatheters, coronary guidewires, drug infusion catheters, esophagealstents, circulatory support systems, angiographic catheters, coronaryand peripheral guidewires, hemodialysis catheters, neurovascular ballooncatheters, tympanostomy vent tubes, cerebro-spinal fluid shunts,defibrillator leads, percutaneous closure devices, drainage tubes,thoracic cavity suction drainage catheters, electrophysiology catheters,stroke therapy catheters, abscess drainage catheters, biliary drainageproducts, dialysis catheters, central venous access catheters, andparental feeding catheters.

Other examples of medical devices suitable for the present inventioninclude, but are not limited to implantable vascular access ports, bloodstorage bags, blood tubing, central venous catheters, arterialcatheters, vascular grafts, intraaortic balloon pumps, cardiovascularsutures, total artificial hearts and ventricular assist pumps,extracorporeal devices such as blood oxygenators, blood filters,hemodialysis units, hemoperfusion units, plasmapheresis units, andartificial organs. It is noted that in other embodiments of the presentinvention, the CMP conjugates of the present invention may also beadhered to the medical device by means other than coating materials,such as adhesives or compression.

In one embodiment, a polymeric solution is applied to the surface of adevice, and a CMP conjugate is applied subsequently. The polymericsolution comprising the CMP is allowed to dry, cure and/or polymerizethereby fixing the CMP conjugate to the polymeric material coating. Inanother embodiment, the CMP is allowed to associate with the polymerprior to, during or following application of the CMP polymer conjugateto the device. In still other embodiments, the CMP conjugate includes atherapeutic agent suitable for delivery from a CMP polymer coating.

Any suitable administration method know in the art may be utilized toadminister the CMP conjugate to the surface. For example, the CMPconjugate may be administered to the surface by press rolling thepolymer coated surface in the CMP conjugate, spraying the CMP conjugateonto the device, or gently heating (e.g., below the transition meltingtemperature) the polymer coating in the presence of a CMP conjugate,such that the heating facilitates the incorporation of the CMP conjugateinto the polymer. Typically, such heating is to a temperature sufficientto denature the CMP.

The polymeric materials with biocompatible surfaces may be utilized forvarious medical applications including, but not limited to, drugdelivery devices for the controlled release of pharmacologically activeagents, including drug delivery patches, encapsulated or coated stentdevices, vessels, tubular grafts, vascular grafts, wound healingdevices, including protein matrix suture material and meshes,skin/bone/tissue grafts, adhesion prevention barriers, cell scaffolding,medical device coatings/films and other biocompatible implants.

Wound Healing Devices

The present invention also provides wound healing compositions thatutilize a polymeric material comprising a CMP conjugate. The woundhealing devices may be configured by fixing a CMP conjugate to a polymer(e.g., a collagen film or PEODA) and forming the CMP conjugatecontaining polymer into a shape and size sufficient to accommodate thewound being treated. Such wound healing devices are desirably producedin whatever shape and size is necessary to provide optimum treatment tothe wound. These devices can be produced in forms that include, but arenot limited to, plugs, meshes, strips, sutures, or any other form ableto accommodate and assist in the repair of a wound. The damaged portionsof the patient that may be treated with a devices made of the particlesof the present invention include, but are not limited to, skin, tissue(nerve, muscle, cartilage, brain, spinal cord, heart, lung, etc.) andbone. Other similar devices are administered to assist in the treatmentrepair and remodeling of a damaged tissue, bone, or cartilage. Ifdesired, the wound healing device comprises a matrix containing one ormore cells. For some applications, it is desirable for the device to beincorporated into an existing tissue to facilitate wound repair. Forother applications, it is desirable for the device to degrade over thecourse of days, weeks, or months.

If desired the CMP is conjugated to a therapeutic agents and the CMPconjugate is delivered to a wound using a polymeric material to form adelivery system. Preferably, the polymer contains an effective amount ofa CMP conjugate that includes a dosage of the chemical orpharmaceutically active component. An adhesive or other adhering meansmay be applied to the outer edges of the polymeric material to hold thepatch in position during the delivery of the chemical orpharmaceutically active component. This polymeric delivery systemprovides for the systematic and/or locally administration of a desiredamount of a therapeutic agent.

Other embodiments of the present invention include wound-healing devicesconfigured and produced as polymeric material biological fasteners, suchas threads, sutures and woven sheets. Threads and sutures comprisingvarious embodiments of the polymeric material provide a biocompatiblefastening and suturing function for temporarily treating and sealing anopen wound. Additionally, the biological fasteners may includepharmacologically active agents that may assist in the healing andremodeling of the tissue within and around the wound.

In other embodiments, the CMP conjugate is administered directly to aninjured area. The CMP conjugate is administered by sprinkling, packing,implanting, inserting or applying or by any other administration meansto open wounds on the body.

Hemostatic Applications

The invention further provides hemostatic matrices (e.g., hemostaticsponges) that contain a polymer, such as collagen, and a CMP or CMPconjugate capable of absorbing bodily fluids (e.g. blood). Such matricesgenerally comprise porous compositions formed from a suitablebiocompatible matrix. Suitable biocompatible matrix materials includenaturally-occurring polymers, such as collagen, and/or syntheticpolymers. In general, sponge matrices can be formed by providing aliquid solution or suspension of a matrix-forming material, and causingthe material to form a porous three-dimensionally stable structure. Inone embodiment, a collagen solution is prepared. Collagen is derivedfrom a natural source (e.g., bovine, porcine, human) or issynthetically-derived. The collagen is typically digested to form acollagen solution. Such digestion is usually carried out under acidicconditions. Optionally, enzymatic digestion may be utilized using knownenzymes for this purpose such as pepsin, trypsin, and/or papain. Afterdigestion, the enzymes are removed using standard methods. Additionalinformation relating to collagenous matrix materials and theirpreparation, is described in U.S. Pat. Nos. 4,511,653, 4,902,508,4,956,178, 5,554,389, and 6,099,567, and International Publication Nos.WO9825637 and WO9822158, each of which is hereby incorporated herein byreference in its entirety.

A CMP or CMP conjugate is incubated with the collagen prior to, during,or after polymerization, such that the CMP or CMP conjugate isincorporated into the collagen. This incubation is typically carried outat a temperature that is above the melting point of the CMP asdetermined using methods described herein. If desired, the collagensolution is heated gently (e.g., at or below the transition meltingtemperature of the CMP) to destabilize the collagen and to facilitateincorporation of the CMP. Preferably, the heating is below the levelrequired to denature the collagen. If desired, the resulting collagensolution is crosslinked. Such crosslinking may be achieved using CMPs.Alternatively, conventional crosslinking agents are used, such asglutaraldehyde, formaldehyde, carbodiimides, UV irradiation, or othercrosslinking agents. In one embodiment, a polyepoxide crosslinker isused, such as polyglycidyl ether (e.g., ethylene glycol diglycidylether). Typically, polyglycidyl ethers or polyepoxide compounds impartpolar groups and a hydrophilic character to the resulting matrix.Preferably, the resulting matrix is wettable and provides for rapidhydration and expansion.

Sponge matrix materials of the invention will advantageously be highlyexpandable when wetted. Preferably, the sponge has the capacity toexpand at least 100%, 200%, 300%, 500%, or 1000% by volume when wettedto saturation with deionized water. Preferred sponge materials achieverapid volume expansions (e.g., maximum expansion in less than 10 secondsor 5 seconds, when immersed in deionized water) Hemostatic sponges areproduced in any size required for application to a wound. Preferably,the expanded sponge exerts compression on surrounding tissues whenimplanted or delivers an active agent to the implantation site andsurrounding tissue.

Compact, dense sponge matrices of the invention are prepared by firsthydrating a porous sponge matrix, and then compressing and drying thematrix. Drying will be conducted to reduce the liquid (e.g. water)content of the matrix to less than about 5%, 10%, or 20% by weight. Thesponge matrix is stabilized structurally and remains in a highly denseand compacted state until contacted with a liquid susceptible toabsorption by the matrix, for example body fluids. The pores of thematrix are thereby stably retained at a volume substantially reducedfrom their maximum volume, but return to a partially or fully expandedstate when the matrix material is wetted.

For medical use, the compacted or compressed sponge is sterilized usingany suitable means (e.g., radiation). The device is packaged in sterilepackaging for medical use.

Sponge elements or other devices of the invention may also contain oneor more active therapeutic agents. For example, they include agents thatpromote clotting (e.g., thrombin and/or fibrinogen). Alternatively or inaddition, sponge elements or other devices of the invention includegrowth factors that promote tissue growth and healing.

Methods for Constructing Engineered Tissue Scaffolds

Polymeric matrices that comprise a collagen mimetic peptide are usefulfor supporting the survival of a variety of cell types including, butnot limited to, endothelial cells, dendritic cells, stem cells or othermultipotent progenitor cells, skin cells, liver cells, heart cells,kidney cells, pancreatic cells, lung cells, bladder cells, stomachcells, intestinal cells, cells of the urogenital tract, breast cells,skeletal muscle cells, skin cells, bone cells, cartilage cells,keratinocytes, hepatocytes, gastro-intestinal cells, epithelial cells,endothelial cells, mammary cells, skeletal muscle cells, smooth musclecells, parenchymal cells, osteoclasts, or chondrocytes. These cell-typesmay be introduced prior to, during, or after gelation. This introductionmay take place in vitro or in vivo. When the cells are introduced invivo, the introduction may be at the site where implantation is desiredor at a location removed from that site. Exemplary routes ofadministration of the cells include injection (e.g., by catheter) andsurgical implantation.

A polymeric matrix comprising a collagen mimetic peptide can be filledwith cells from virtually any organ. Because many cell-types can beexpanded in vitro, grafts can be made using a limited number of cells(e.g., 100, 500, 1000, 10,000, 100,000, 1,000,000, 10,000,000, or100,000,000), which represent a small percentage (e.g., 0.0001%, 0.001%,0.005%, 0.01%, 0.05%, 0.10%, 1.0%, 2.0%, or 5.0%) of the cells presentin a naturally-occurring tissue or organ. Exemplary cells fororganogenesis include, hepatocytes, myocytes (e.g., cardiac or skeletalmuscle myocytes), keratinocytes, osteocytes, chondrocytes, islet cells,nerve cells, astrocytes, glial cells from the central or peripheralnervous system, preadipocytes derived from fat or breast tissue, andadipocytes. Such cells might be obtained from the intended implantrecipient (an autograft), from a donor (allogeneic graft), or from acell line. One particular advantage of autografts is that the graftedtissue does not induce an immune response because the grafted cells arerecognized as self (Heath et al., Trends Biotechnol, 18: 17-19, 2000).In other embodiments, such cells are obtained from a mammal of adifferent species (e.g., pig or primate).

Cell Isolation

One aspect of the invention pertains to a matrix that may be used topromote the healing of an injured tissue, for replacement of a damagedor absent tissue, or for tissue augmentation. The implanted cells may bederived from the recipient's own tissue, derived from a differentindividual of the same species, or derived from a mammalian species thatis different from the recipient (e.g., pig or primate). Cells can beisolated from a number of sources, for example, from biopsies orautopsies using standard methods. The isolated cells are preferablyautologous cells obtained by biopsy from the subject. The cells from abiopsy can be expanded in culture. Cells from relatives or other donorsof the same species can also be used with appropriate immunosuppression.Methods for the isolation and culture of cells are discussed in Fauza etal. (J. Ped. Surg. 33, 7-12, 1998).

Cells are isolated using techniques known to those skilled in the art.For example, a selected tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withdigestive enzymes (e.g., trypsin, chymotrypsin, collagenase, elastase,hyaluronidase, DNase, pronase, and dispase). Mechanical disruption canbe accomplished by scraping the surface of the organ, the use ofgrinders, blenders, sieves, homogenizers, pressure cells, or sonicators.For a review of tissue disruption techniques, see Freshney, (Culture ofAnimal Cells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., NewYork, Ch. 9, pp. 107-126, 1987) Preferred cell types include, withoutlimitation, chondrocytes, endothelial cells, osteoblasts, and nervecells. Once the tissue has been reduced to a suspension of individualcells, the suspension can be fractionated into subpopulations. This maybe accomplished using standard techniques (e.g., cloning and positiveselection of specific cell types or negative selection, i.e., thedestruction of unwanted cells). Selection techniques include separationbased upon differential cell agglutination in a mixed cell population,freeze-thaw procedures, differential adherence properties of the cellsin the mixed population, filtration, conventional and zonalcentrifugation, unit gravity separation, countercurrent distribution,electrophoresis and fluorescence-activated cell sorting. For a review ofclonal selection and cell separation techniques, see Freshney, Cultureof Animal Cells, A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc.,New York, Ch. 11 and 12, pp. 137-168, 1987).

Cell fractionation may be useful when the donor has a disease, such ascancer. Isolated cells can be cultured in vitro to increase the numberof cells available for transplantation. The use of allogenic cells, andmore preferably autologous cells, is preferred to prevent tissuerejection. However, if an immunological response does occur in thesubject after implantation of the engineered organ, the subject may betreated with immunosuppressive agents, such as cyclosporin or FK506, toreduce the likelihood of rejection.

Isolated cells may be transfected. Useful genetic material may be, forexample, genetic sequences that are capable of reducing or eliminatingan immune response in the host. For example, the expression of cellsurface antigens such as class I and class II histocompatibilityantigens may be suppressed. This may allow the transplanted cells tohave reduced chance of rejection by the host. In addition, transfectioncould also be used for gene delivery. The cell-substrate construct cancarry genetic information required for the long-term survival of thehost or the artificial organ or for detecting or monitoring the cells.In one example, the implanted cell or cells are genetically modified toexpress a bioactive molecule that promotes cell growth or survival. Inanother example, the cell or cells are genetically modified to expressesa fluorescent protein marker. Exemplary markers include GFP, EGFP, BFP,CFP, YFP, and RFP. The cell-substrate construct can also carry geneticinformation required for promoting or maintaining angiogenesis.Transfection may be used for transient gene expression or stable geneexpression by incorporation of the gene into the host cell.

Isolated cells can be normal or genetically-engineered to provideadditional or normal function. Methods for genetically engineering cellswith viral vectors such as retroviral vectors or other methods known tothose skilled in the art can be used. These include using expressionvectors which transport and express nucleic acid molecules in the cells(see, for example, Goeddel et al., (Gene Expression Technology: Methodsin Enzymology 185, AcademiPress, San Diego, Calif., 1990). Vector DNA isintroduced into prokaryotic oreukaryotic cells via conventionaltransformation or transfection techniques. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

Methods for Repairing Damaged Tissues and Organs

The invention features methods of repairing diseased or damaged tissuesand organs. Cells are administered to a damaged or diseased tissue ororgan. These methods may stabilize a damaged tissue or organ in apatient on a transplantation waiting list; or the methods may repair adamaged or diseased tissue or organ, thereby obviating the need fortransplantation. Methods for repairing damaged tissue or organs may becarried out either in vitro, in vivo, or ex vivo.

Methods for Producing Engineered Tissues or Organs

The invention features methods of producing an engineered replacementtissue. Cells are preferably cultured in the presence of a matrix thatcontains a CMP or CMP conjugate. If desired, a CMP conjugate is used tomodulate the adhesive properties of the matrix. For some applications,the CMP is conjugated to a PEG, such that the CMP PEG conjugate isdesigned to repel the adhesion of a cell. In another embodiment, the CMPconjugate is designed to promote cell adhesion, thereby promotingincorporation of the matrix (e.g., a synthetic polymer based matrix,decellularized skin or other tissue source; collagen or otherextracellular matrix gel) into an existing tissue as described herein.Methods for producing an engineered tissue or organ may be carried outeither in vitro, in vivo, or ex vivo. It is also contemplated thatmatrices of the invention comprising cells are administered to a mammalto treat damage or deficiency of cells in an organ, muscle, or otherbody structure, or to form an organ, muscle, or other body structure.Desirable organs include the bladder, brain, nervous tissue, glialtissue, esophagus, fallopian tube, heart, pancreas, intestines,gallbladder, kidney, liver, lung, ovaries, prostate, spinal cord,spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract,ureter, urethra, uterus, breast, skeletal muscle, skin, bone, andcartilage.

Engineered Bone or Cartilage

Exemplary transplantation methods of the present invention includerepairing or replacing bone or cartilaginous tissue, such as articularcartilage. Traditional bone or cartilage tissue engineering methods canbe improved by administering chondrocytes in a matrix comprisingcollagen mimetic peptides to the damaged or diseased bone or cartilagein vivo or to a bone or cartilage transplant tissue before, during, orafter the transplant tissue is administered to a mammal. Traditionalbone and cartilaginous tissue reconstruction methods are described, forexample, in U.S. Pat. Nos. 6,197,061; 6,197,586; 6,228,117; 6,419,702;and 6,451,060. Engineered bone is useful for the treatment of a varietyof diseases or disorders, including arthritis, cancer, congenitaldefects of bone or cartilage such as worn or torn cartilage in jointlinings (e.g., knee joint, hip joint, and temporomandibular joint) andtrauma. It is known that connective-tissue cells, including fibroblasts,cartilage cells, and bone cells, can undergo radical changes ofcharacter. A great variety of materials are useful as matrices for thispurpose. For example, materials such as PEODA, collagen gels, poly (D,L-lactide-co-glycolide (PLGA) fiber matrices, polyglactin fibers,calcium alginate gels, polyglycolic acid (PGA) meshes, and otherpolyesters such as poly-(L-lactic acid) (PLLA) and polyanhydrides areamong those suggested. Matrices can include materials that arenon-biodegradable or biodegradable. Desirably, biodegradable materialswill degrade over a time period of less than a year, more preferablyless than six months.

Methods for treating connective tissue disorders using engineeredcartilaginous or connective tissues are described, for example, in U.S.Pat. Nos. 5,226,914; 5,041,138; 5,368,858; 5,632,745; 6,451,060;6,197,586; and 6,197,061. Surgical procedures related to bone tissuedeficiencies vary from joint replacement or bone grafting tomaxillo-facial reconstructive surgery. Such methods are known to theskilled artisan.

Engineered Soft Tissue

Traditional methods of soft tissue reconstruction, as described in U.S.Pat. No. 5,716,404, can be improved by administering cells of theinvention in a matrix comprising collagen mimetic peptides to the softtissue to be transplanted. For example, engineered soft tissue is usefulfor cosmetic surgery or for reconstruction of the breast, face, or otherbody part after cancer surgery or trauma. For soft tissuereconstruction, the matrix, which is mixed with cells may form ahydrogel.

Engineered Tissue and Organ Transplantation

A donor organ, donor cell, engineered tissue, or engineered organ istransplanted into a patient (e.g., a human or mammal) for the treatmentor stabilization of a condition, disease, or disorder using standardmethods known to the skilled artisan. Methods for three-dimensionalskeletal muscle tissue-engineering are described by Saxena et al.,(Biomed. Mater. Eng. 11 (4): 275-281, 2001).

Corneal Shields

Corneal shields of the invention are ophthalmic lens that containcollagen in association with a collagen mimetic peptide Collagen is amajor component of the white sclera and the clear cornea. Natural orsynthetic collagens are shaped into a contact lens that can be placed onthe surface of the eye. The collagen shield provides a protectiveenvironment that promotes the healing of surgical and traumatic woundsto the eye. Such shields are useful as ophthalmic dressings and asophthalmic drug delivery devices. Drugs or diagnostic agents which canbe administered include antibiotics, such as beta-lactam antibiotics,such as cefoxitin, n-formamidoylthienamycin and other thienamycinderivatives, tetracyclines, chloramphenicol, neomycin, carbenicillin,colistin, penicillin G, polymyxin B, vancomycin, cefazolin,cephaloridine, chibrorifamycin, gramicidin, bacitracin and sulfonamides;anti-inflammatories, such as cortisone, hydrocortisone, hydrocortisoneacetate, betamethasone, dexamethasone, dexamethasone sodium phosphate,prednisone, methylprednisolone, medrysone, fluorometholone,prednisolone, prednisolone sodium phosphate, triamcinolone,indainethacin, sulindac. Desirably, the corneal shield comprises aphysiologically acceptable vehicle having a buffered pH and hypoosmotic,hyperosmotic, or isoosmotic characteristics. Typically, the pH andosmolality of the ophthalmic delivery device is matched to the pH andosmolality of the eye. In some embodiments, the corneal shield issubject to degradation over time. Such degradation is typicallyaccomplished via a naturally occurring enzyme present in tears.

Delivery of Collagen Mimetic Peptide Therapeutics

Collagen mimetic peptide therapeutics include cell containing matricesas well as collagen mimetic peptide conjugates. These therapeutics canbe delivered by any method known to the skilled artisan. In oneapproach, a CMP conjugate is administered via an intravenous catheterthat is inserted into a blood vessel and guided through am artery orvein to a desired location. For example, a CMP conjugate that includesan anti-thrombotic is administered via a catheter directly to a clot.The end of the catheter may be placed in the vessels leading to thebrain, lung, heart, arm, or leg depending upon the location of the clot.In another approach, a liquid solution containing cells and CMP-polymerconjugates is injected into a desired site or is surgically implanted.The liquid is then polymerized in vivo. Alternatively, the CMP-polymerconjugate is polymerized in vitro and subsequently administered.

The present invention provides methods of treating diseases and/ordisorders or symptoms thereof which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising a collagen mimetic peptide of a formulae herein to a subject(e.g., a mammal such as a human). Thus, one embodiment is a method oftreating a subject suffering from or susceptible to a disease ordisorder that requires targeting of a therapeutic composition to a sitecomprising collagen or a symptom thereof. The method includes the stepof administering to the mammal a therapeutic amount of a compound hereinsufficient to treat the disease or disorder or symptom thereof, underconditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a disease, disorder, or symptomthereof. Determination of those subjects “at risk” can be made by anyobjective or subjective determination by a diagnostic test or opinion ofa subject or health care provider (e.g., genetic test, enzyme or proteinmarker, Marker (as defined herein), family history, and the like). Thecompounds herein may be also used in the treatment of any otherdisorders in which it is desirable to target a tissue comprisingcollagen may be implicated.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof, in which the subjecthas been administered a therapeutic amount of a compound hereinsufficient to treat the disease or symptoms thereof. The level of Markerdetermined in the method can be compared to known levels of Marker ineither healthy normal controls or in other afflicted patients toestablish the subject's disease status. In preferred embodiments, asecond level of Marker in the subject is determined at a time pointlater than the determination of the first level, and the two levels arecompared to monitor the course of disease or the efficacy of thetherapy. In certain preferred embodiments, a pre-treatment level ofMarker in the subject is determined prior to beginning treatmentaccording to this invention; this pre-treatment level of Marker can thenbe compared to the level of Marker in the subject after the treatmentcommences, to determine the efficacy of the treatment.

Methods for Evaluating Therapeutic Efficacy

Methods of the invention are useful for treating or stabilizing in apatient (e.g., a human or mammal) a condition, disease, or disorderaffecting a tissue or organ. Therapeutic efficacy is optionally assayedby measuring, for example, the biological function of the treated ortransplanted organ (e.g., bladder, bone, brain, breast, cartilage,esophagus, fallopian tube, heart, pancreas, intestines, gallbladder,kidney, liver, lung, nervous tissue, ovaries, prostate, skeletal muscle,skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,ureter, urethra, urogenital tract, and uterus). Such methods arestandard in the art and exemplary methods follow. Preferably, atransplantation method of the present invention, increases thebiological function of a tissue or organ by at least 5%, 10%, 20%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or even by as much as 300%,400%, or 500%. In addition, the therapeutic efficacy of the methods ofthe invention can optionally be assayed by measuring an increase in cellnumber in the treated or transplanted tissue or organ as compared to acorresponding control tissue or organ (e.g., a tissue or organ that didnot receive treatment). Preferably cell number in a tissue or organ isincreased by at least 5%, 10%, 20%, 40%, 60%, 80%, 100%, 150%, or 200%relative to a corresponding tissue or organ. Methods for assaying cellproliferation are known to the skilled artisan and are described in(Bonifacino et al., Current Protocols in Cell Biology Loose-leaf, JohnWiley and Sons, Inc., San Francisco, Calif.).)

Screening Assays

As described herein, the present invention provides for the delivery oftherapeutic agents to cells, tissues, or organs in vitro or in vivo. Theinvention is based in part on the discovery that therapeutic agents canbe conjugated to a collagen mimetic peptide. Collagen is then contactedwith the collagen mimetic peptide conjugate, such that the collagenmimetic peptide conjugate physically associates with collagen. Based inpart on this discovery, compositions of the invention are useful for thehigh-throughput low-cost screening of compounds conjugated to CMP.Desired CMP conjugates physically interact with collagen (e.g., bindwith high affinity) without disrupting collagen structure. The CMPconjugate thereby modulates a biological function of a cell, tissue, ororgan. Collagen, or tissues or cells comprising collagen are treatedwith a collagen mimetic peptide conjugate and are subsequently comparedto untreated control samples to identify therapeutic CMP conjugates thatbind collagen with high affinity without disrupting collagen structureand/or enhance a biological function of the cell, tissue, or organ. Anynumber of methods are available for carrying out screening assays toidentify such compounds.

In one working example, candidate compounds are added at varyingconcentrations to the culture medium of cultured cells. Any desiredbiological function is then measured using standard methods. Thebiological function in the presence of the candidate compound iscompared to the level measured in a control culture medium lacking thecandidate molecule. A compound that enhances cell function is considereduseful in the invention; such a candidate compound may be used, forexample, as a therapeutic to prevent, delay, ameliorate, stabilize, ortreat a disease described herein (e.g., atherosclerosis, thrombosis,inflammation, tissue damage). In other embodiments, the candidatecompound prevents, delays, ameliorates, stabilizes, or treats a diseaseor disorder described herein. Such therapeutic compounds are useful invivo as well as ex vivo.

In one working example, CMP conjugates are screened for those thatspecifically bind to collagen. The efficacy of such a candidate compoundis dependent upon its ability to interact with collagen, or withfunctional equivalents thereof. Such an interaction can be readilyassayed using any number of standard binding techniques and functionalassays (e.g., those described in herein).

In one example, a CMP conjugate that binds to a collagen is identifiedusing a chromatography-based technique. For example, collagen isimmobilized on a column. A solution comprising a CMP conjugate is thenpassed through the column, and a CMP conjugate is identified on thebasis of its ability to bind to the collagen and be immobilized on thecolumn. To isolate the CMP conjugate, the column is washed to removenon-specifically bound molecules, and the CMP conjugate of interest isthen released from the column and collected. Similar methods may be usedto isolate a CMP conjugate bound to a collagen microarray comprising anycollagen type desired (e.g., collagen types 1-29). A CMP conjugateidentified using such methods is assayed for an effect on the biologicalfunction of a cell, tissue, or organ as described herein.

In another example, the CMP conjugate, e.g., the substrate, is coupledto a radioisotope or enzymatic label such that binding of the compound,e.g., the substrate, to collagen can be determined by detecting thelabeled compound, e.g., substrate, in a complex. For example, compoundscan be labeled with 125I, ³⁵S, ¹⁴C, or ³H, either directly orindirectly, and the radioisotope detected by direct counting ofradioemmission or by scintillation counting. Alternatively, compoundscan be enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In yet another embodiment, a cell-free assay is provided in which a CMPconjugate or a biologically active portion thereof is contacted with acollagen and the ability of the CMP conjugate to bind to the collagenthereof is evaluated.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of a CMP conjugate tobind to a collagen can be accomplished using real-time BiomolecularInteraction Analysis (BIA) (see, e.g., Sjolander, S, and Urbaniczky, C.,Anal. Chem. 63:2338-2345, 1991; and Szabo et al., Curr. Opin. Struct.Biol. 5:699-705, 1995). “Surface plasmon resonance” or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the mass at the binding surface(indicative of a binding event) result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)), resulting in a detectable signal that can beused as an indication of real-time reactions between biologicalmolecules.

It may be desirable to immobilize either the CMP conjugate or thecollagen target to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a CMP conjugate to a collagen, orinteraction of a CMP conjugate with a target collagen in the presenceand absence of a CMP conjugate, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., Trends Biochem Sci 18:284-7, 1993); chromatography(gel filtration chromatography, ion-exchange chromatography);electrophoresis and immunoprecipitation (see, for example, Ausubel, F.et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley:New York). Such resins and chromatographic techniques are known to oneskilled in the art (see, e.g., Heegaard, N. H., J Mol Recognit 11:141-8,1998; Hage, D. S., and Tweed, S. A., J Chromatogr B Biomed Sci Appl.699:499-525, 1997). Further, fluorescence energy transfer may also beconveniently utilized, as described herein, to detect binding withoutfurther purification of the complex from solution.

One skilled in the art appreciates that the effects of a CMP conjugateon biological activity are typically compared to the biological activityin the absence of the CMP conjugate. Thus, the screening methods includecomparing the value of a cell modulated by a candidate CMP conjugate toa reference value of an untreated control cell. Changes in tissue ororgan morphology further comprise values and/or profiles that can beassayed by methods of the invention by any method known in the art.

Test Compounds and Extracts

In general, therapeutic compounds suitable for coupling to CMP areidentified from large libraries of both natural product or synthetic (orsemi-synthetic) extracts or chemical libraries or from polypeptide ornucleic acid libraries, according to methods known in the art. Thoseskilled in the field of drug discovery and development will understandthat the precise source of test extracts or compounds is not critical tothe screening procedure(s) of the invention. Compounds used in screensmay include known compounds (for example, known therapeutics used forother diseases or disorders). Alternatively, virtually any number ofunknown chemical extracts or compounds can be screened using the methodsdescribed herein. Examples of such extracts or compounds include, butare not limited to, plant-, fungal-, prokaryotic- or animal-basedextracts, fermentation broths, and synthetic compounds, as well asmodification of existing compounds.

Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds tobe used as candidate compounds can be synthesized from readily availablestarting materials using standard synthetic techniques and methodologiesknown to those of ordinary skill in the art. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing the compounds identified by themethods described herein are known in the art and include, for example,those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422,1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al.,Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired,any library or compound is readily modified using standard chemical,physical, or biochemical methods.

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S.Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids(Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage(Scott and Smith, Science 249:386-390, 1990; Devlin, Science249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382,1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their activity should be employed wheneverpossible.

When a crude extract is identified as containing a compound of interest,further fractionation of the positive lead extract is necessary toisolate chemical constituents responsible for the observed effect. Thus,the goal of the extraction, fractionation, and purification process isthe careful characterization and identification of a chemical entitywithin the crude extract that achieves a desired biological effect.Methods of fractionation and purification of such heterogenous extractsare known in the art.

Small molecules of the invention preferably have a molecular weightbelow 2,000 daltons, more preferably between 300 and 1,000 daltons, andmost preferably between 400 and 700 daltons. It is preferred that thesesmall molecules are organic molecules.

Kits

The invention provides diagnostic and therapeutic kits that include CMPor CMP conjugates capable of interacting with collagen in vitro or invivo. In one embodiment, the kit includes a diagnostic compositioncontaining a detectable CMP conjugate (e.g., CMP conjugated to ananoparticle, a detectable label, or a contrast reagent). In otherembodiments, the kit contains a therapeutic device, such as a woundhealing device, hemostatic sponge, or drug delivery device containing aCMP conjugate and a polymer.

In some embodiments, the kit comprises a sterile container whichcontains a CMP conjugate or a CMP conjugate and a polymer; suchcontainers can be boxes, ampules, bottles, vials, tubes, bags, pouches,blister-packs, or other suitable container forms known in the art. Suchcontainers can be made of plastic, glass, laminated paper, metal foil,or other materials suitable for holding medicaments.

If desired a CMP conjugate of the invention is provided together withinstructions for using it in a diagnostic or therapeutic methoddescribed herein. The instructions will generally include informationabout the use of the composition for the diagnosis or treatment of adisease in a subject in need thereof. The instructions may be printeddirectly on the container (when present), or as a label applied to thecontainer, or as a separate sheet, pamphlet, card, or folder supplied inor with the container.

EXAMPLES Example 1 Methods of Characterizing a CMP or CMP Conjugate

To determine whether the propensity of CMPs to form collagen-like triplehelices enables it to bind to partially denatured collagen byassociating with disentangled domains of the collagen molecules, CMP and5-carboxy fluorescein (5CF) labeled CMP derivatives were prepared asshown in Table 1 (Table 1:1 and 2, respectively), which provides thesequence and transition melting temperature (T_(m)) of peptides 1-5, asdetermined by circular dichroism spectroscopy (FIGS. 1-3).

TABLE 1 Transition Temperatures of Collagen Mimetic Peptide DerivativesDetermined by Circular Dichroism Spectroscopy^(a). Compound Sequence SEQID NO: Tm 1 -(ProHypGly)10- 36 69° C. 2 5CF-Gly3-(ProHypGly)10- 37 75°C.  3^(b) 5CF-Gly3-randomPro10Hyp10Gly10- 38 — 4 5CF-Gly3-(ProHypGly)6-39 25° C.  5^(c) mPEG2000-Gly3-(ProHypGly)7- 6 29° C. ^(a)measured in57.5 μM acetic acid solution.^(b)5CF-GGGGPPP^(H)P^(H)GPGGGPP^(H)PP^(H)GP^(H)GPP^(H)PGP^(H)P^(H)PGGP^(H)P^(H)PP(SEQ ID NO: 38), (P^(H):Hyp). ^(c)mPEG₂₀₀₀: CH₃O—(CH₂—CH₂—O)_(n)—OH,2250 Da

In addition, peptide 3 was synthesized having the same amino acidcomposition as peptide 2, but the Pro, Hyp, Gly sequence was scrambled,rendering peptide 3 non-helicogenic. Three consecutive glycines wereinserted as a spacer between the fluorescence tag and the CMP. Attaching5CF and three glycines to 1 elevated its melting temperature from 69° C.to 75° C. (Table 1). This is due in part to the hydrophobic nature ofthe fluorescence tag. Attachment of a hydrophobic fatty acid to the CMPwas previously shown to stabilize the triple helix elevating its meltingtemperature.⁵ As expected, no melting behavior was observed for compound3.

The binding of CMP to natural collagen (acid soluble, bovine type I) ordenatured collagen (gelatin) was demonstrated by treating collagen filmswith solutions of the fluorescently labeled CMP, rinsing, and measuringthe fluorescence intensity of the exposed film. The transition meltingtemperature of collagen film as determined by circular dichroismspectroscopy is shown at FIG. 4. To the collagen-coated cell culturewells (at room temperature) was added a solution of 2 which waspre-equilibrated at either 25° C. or 80° C. After three hours ofincubation at room temperature, the collagen films were washed withbuffer solution and observed by fluorescence microscope. As controlsamples, 5CF, fluorescein isothiocyanate-dextran (FITC-Dextran), andpeptide 3 were used to treat collagen films under identical experimentalconditions. All control samples exhibited negligible affinity towardcollagen film evidenced by low fluorescence intensity at bothexperimental conditions (FIG. 5A: Group a and b). Collagen film treatedwith peptide 2 at 25° C. also showed negligible fluorescence. Incontrast, the collagen film treated with peptide 2 that waspre-equilibrated at 80° C., the temperature above peptide 2's meltingtransition temperature (75° C.), exhibited strong fluorescence. Similarresults were obtained when gelatin films were used as a substrate (FIG.5A: Group c). In addition, the helical content of CMP treated collagenfilm was 3.5 times higher than that of the film treated with a blanksolution (see support information). These results suggested that peptide2 tightly attached to partially denatured collagen when it is introducedas a single strand and that its ability to assemble into triple helix isrelevant for the attachment.

In order to understand the effect of collagen film denaturation in theCMP binding process, a shorter CMP derivative was synthesized, peptide4, with a melting temperature (25° C.) well below that of the collagenfilm. Little binding was observed when a peptide 4 solution at 10° C.was used to treat the collagen film (FIG. 5B). Treatment with the samesolution pre-equilibrated at 30° C., the temperature above 4's meltingtemperature but below the denaturation temperature of collagen film (37°C.), induced more than three-fold increase in CMP attachment compared tothat of the 10° C. solution. In addition, the modified collagen fiberretained its native banding texture when investigated by transmissionelectron microscopy (FIG. 6). Hotter solutions (60° C. and 80° C.) whichdenature the collagen film during the treatment produced collagen filmswith slightly higher CMP content (approximately 20% increase from thatof the 30° C. solution). These results indicated that the CMP meltinginto monomers, but not collagen denaturation supported CMPimmobilization.

The observed adhesion arises from a strand exchange reaction and triplehelix association between CMP and the collagen. It is interesting tonote that a number of researchers have proposed existence of thermallylabile domains within the type I collagen sequence which may serve aspotential sites for the presumed strand exchange reaction.⁶ Within thecollagen family a class of collagen known as Fibril Associated Collagenswith Interrupted Triple-helices exists (FACITs)²²⁻²⁴. FACITs do not formfibrous structure by themselves but are always found as individualcollagen molecules decorating the surface of collagen fibers. Thefindings reported herein do not indicate if CMP is binding to thethermally labile domains, or if the binding event mimics that of a FACITprotein.

To evaluate the potential of the new modification technique in tissueengineering, poly(ethyleneglycol)₂₀₀₀-CMP conjugate polymer^(25,26)(Table 1: 5) was prepared. This modified collagen was designed to reducethe adhesiveness of collagen to cells.

The melting temperature of 5 was determined to be 29° C. which is 7° C.lower than the melting temperature of (ProHypGly)₇ (SEQ ID NO: 40)(Table 1).²⁷ Here, the hydrophilic and bulky PEG group seems todestabilize the triple helix in water. A solution containing 5 at 45° C.was added to the collagen coated culture plate (prepared as above) andhuman fibroblasts or breast epithelial cells were seeded and incubatedfor three days at 37° C. Homogenous distribution of fibroblasts is seenon the collagen film that was treated with mPEG₂₀₀₀ (Control sample,FIG. 7A). In contrast, areas of collagen films treated with 5 are almostdevoid of fibroblasts (FIG. 7B) and epithelial cells (FIG. 7C). Thisexperiment demonstrates that the adhesive properties of prefabricatedcollagen film can be readily modified by the simple action of deliveringCMP conjugate solutions to the target area.

These results demonstrate that a prefabricated collagen matrix can bereadily modified by delivering CMP conjugate solutions to a target area(FIG. 8). Such methods are useful for applications where it is desirableto repel cell growth while new tissue structures are organized.

For applications where it is desirable to repel cell growth forprolonged periods (e.g., weeks or months), it is desirable to employCMPs of increased length. In films that were treated withmPEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6), cell growth extending intothe treated area was observed after 3 days in culture (FIGS. 9A, 9B, and9C). This might be due to the release of mPEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇(SEQ ID NO: 6) from the PEG-CMP modified collagen film during incubationat 37° C.

(Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO: 26) with lengthened CMP andPEG, was designed to prolong the anti-adhesive property of pegylated CMPmodified collagen to cells. Its melting temperature is 38° C. in PBSsolution. A delay in invasive cell growth was observed on areas of thecollagen film treated with (Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO:26) (FIG. 10A and FIG. 10B). The treated gel repelled extensive cellgrowth for close to eleven days. After five days in culture, littleinvasion into treated areas of the collagen film is observed. After ninedays in culture only moderate invasion is observed. Extensive invasionis observed after eleven days in culture. It can also be seen from theimages that cells outside the treated area grow vertically beforeinvading the treated area.

To determine whether PEG-CMP derivatives are diffusing out of the gel,fluorescence labeled PEG-CMPs, FL-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ IDNO: 30) and 5CF-Gly₃-(Pro-Hyp-Gly)_(s)-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO: 31),were prepared. The CF-Gly₃-(Pro-Hyp-Gly)₁₀ (SEQ ID NO: 51) derivativesbound collagen film with high affinity. FIG. 11 shows a quantitation ofthe release of fluorescence-labeled CMP derivatives from culture dishand collagen film at 37° C. in PBS solution (pH 7.4). On thefluorescence labeled PEG-CMP modified area of collagen film, the releaseof FL-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 30) from collagen film wasfaster than that of 5CF-Gly₃-(Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀-OH (SEQ ID NO:31). The release of fluorescence-labeled PEG-CMPs derivatives fromcollagen films is shown in (FIG. 11). These results suggest that acollagen matrix or other polymer matrix that incorporates a CMPconjugate (e.g., a CMP conjugated to a biologically active agent, isuseful for the controlled release of that CMP conjugate as describedherein.

These experiments indicate that fibroblast and epithelial cell adhesionto natural collagen can be readily altered by applying CMP-poly(ethyleneglycol) conjugates to pre-fabricated collagen films. These PEG-CMPderivatives include methoxy-PEG₂₀₀₀-Gly₃-(Pro-Hyp-Gly)₇ (SEQ ID NO: 6)and (Pro-Hyp-Gly)₈-Gly₃-PEG₅₀₀₀ (SEQ ID NO: 41). Films treated withthese compounds showed dramatically reduced invasion by fibroblasts orepithelial cells. For applications where prolonged repellant activity isrequired stable CMP-PEG derivatives, such as (Pro-Hyp-Gly)₈ (SEQ ID NO:42), will be utilized. (Pro-Hyp-Gly)₈ (SEQ ID NO: 42) exhibits strongeraffinity for collagen. Also useful in such methods are linear PEG ofsuitable lengths or star-shaped PEG. Such molecules have increasedsurface-shielding effects, and are more effective in protecting theconjugates from cell adhesion and protein adsorption (see FIG. 12). Acomparison of protein and cell repellent properties of grafted linearPEG and star shaped PEG has been described¹³⁻¹⁶. In particular, starshaped PEG-coated glass, titanium and silicon samples inhibit celladhesion¹⁷.

In order to increase the cell repelling activity CMP is conjugated tomulti-armed PEG or to star shaped PEG (MW: 10,000 Da). These moleculesprovide for an increase in the number of collagen binding sites.[AcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈]₄-star shaped PEG (core sequencedisclosed as SEQ ID NO: 27) (MW: 10,000 Da) will be synthesized byconjugation of four NHS activated sites on 4-armed star PEG withAcGly-Gly₂-Lys-Gly₃-(Pro-Hyp-Gly)₈ (SEQ ID NO: 29) (FIG. 12). The amountof PEG-CMP immobilized in the collagen scaffold will be empiricallydetermined. [Carboxyfluorescein-Gly₃-Lys-Gly₃-(Pro-Hyp-Gly)₈]₄-starshaped PEG (core sequence disclosed as SEQ ID NO: 43) will be prepared(FIG. 13). Fluorescence tagged star shaped PEG-CMP that can bequantified and visualized by fluorescence techniques will also beprepared to facilitate detection of star shaped PEG-CMP release. HPLCwill be used to purify the CMP-star shaped PEGs and MALDI-TOF will beused to identify the target products and check for purity.

The ability to control the organization of cells in collagen matrixprovides a new pathway for engineered tissues. Furthermore, the affinitybetween the CMP and collagen could issued to immobilize therapeuticdrugs to collagens in the living tissues, and to biomaterials thatincorporate natural collagens.

Example 2 Use of PEG-CMP for the Organization of Endothelial Cell Growth

Results using PEG-CMP to repel fibrobast cell growth suggest thatPEG-CMP can be used to direct the growth and organization of endothelialcell growth as well, given the similarities that exist between the twocell types. Like fibroblasts, endothelial cells rely on integrin-ECMinteraction for initial binding to the scaffolds. To determine theeffects of PEG-CMP collagen gels on proliferation, tubulogenesis, andcapillary sprouting collagen (type I) films are treated with PEG-CMP ofvarying concentrations. The amount and pattern of immobilized PEG-CMPpresent in the collagen film is determined using fluororescienlabeled-PEG. Endothelial cells are then plated on PEG-CMP modifiedcollagen and cell attachment, morphology, and cytoskeletal organizationis assessed to determine the effect of CMP-PEG modified collagen on cellattachment pattern and cell proliferation. Methods of characterizingcytoskeletal organization include staining with Oregon-Green conjugatedphalloidin.

To determine the effects of PEG-CMP on the growth and organization ofendothelial cells in three dimensional collagen gel, endothelial cellsare cultured in PEG-CMP modified three dimensional collagen gels. On atwo dimensional collagen film the PEG-CMP inhibits fibroblastattachment. Without wishing to be bound by theory, this effect is likelydue to the PEG blocking the ECM-integrin interaction. To determinewhether similar effects are observed in three dimensional ECMs, a threedimensional collagen gel containing varying concentrations of PEG-CMP isprepared by adding PEG-CMP to a collagen solution prior to gelation.Cell morphology is analysed to assess the efficacy of PEG-CMP inregulating cell growth, proliferation, tubulogenesis, and capillarysprouting in a three dimensional collagen gel. Samples of the gel areeasily fixed and fluorescently labeled using actin and nuclearimmunostaining. One end of the collagen gel is immersed in fluorosceinlabeled PEG-CMP solution to create a detectable PEG gradient along thelength of the collagen gel. Cell morphology is visualized by actin andnuclear immunostaining, and evaluated using fluorescence microscope,which also provides for visualization of the PEG gradient.

In another approach, CMP-PEG is injected into the collagen gel viamicrosyringe. This injection is performed at the final stage of gelformation thus minimizing damage to the collagen matrix. Thedistribution of immobilized fluororescein labeled PEG-CMP is visualizedand quantified by fluorescence microscope and the morphology ofendothelial cells and capillary distribution is evaluated with respectto the local concentration of PEG-CMP.

Example 3 Tissue Fixation Using (CMP)_(n) Conjugated-HomoMultifunctional PEG as a Cross-Linker

Present methods for tissue fixation use crosslinking reagents, such asformaldehyde, glutaraldehyde (GA), expoxy compounds, carbodiimide,proanthocyanidin and reactive multifunctional PEGs^(18,19). The use ofsuch reagents reduces the antigenicity of many proteins and increasestheir resistance to enzymatic degradation when tissues treated withthese reagents are implanted^(20,21)]. Additional drawbacks to the useof such agents includes their toxicity, unmanageable crosslinking rates,and instability. The present invention provides a a collagencrosslinking reagent that exhibits low cytotoxicity and can formbiocompatible crosslinked products. CMPs conjugated onto multi-armed PEGcompounds mediate physical binding to collagen and can serve ascrosslinking reagents. CMP conjugated with homo-bifunctional orhomo-tetrafunctional PEG, which are commercially available from NEKTAR(San Carlos, Calif.) (such as NHS or thiol) are prepared and purified byHPLC. The reaction products are characterized using MALDI-TOF and CDmeasurements and rheometry, tensile gel swelling analyses and geldegradation analyses using collagenase are carried out on CMPs-multiarmed PEG conjugate cross-linked collagen gels and fixed tissues.

Example 4 CMP Functionalized Gold Nanoparticles

CMP, (Pro-Hyp-Gly)x (Hyp=hydroxyproline), binds to type I collagenmolecules through a process involving both strand invasion andtriple-helix assembly. In an effort to visualize this interaction incollagen fibers, CMP functionalized gold nanoparticles (NPs) wereprepared to use as a transmission electron microscopy marker. The CMPfunctionalized gold NPs were highly stable in aqueous solution andexhibited a preferential affinity to the gap regions of intact type Icollagen fibers.

The triple helical structure of collagen and CMP bears similarity instructure to the DNA double helix. Both collagen and DNA are composed oflong coaxial multiplex helices that are held together by inter-chainhydrogen bonds. They exhibit reversible melting transitions that reflectthe stability and strength of the helix assembly. In DNA, strandinvasion by short DNAs or peptide nucleic acids is well documented inthe literature.²⁸ Although collagens are known to incorporate thermallyunstable domains where small segments of the triple helix are thought tobe partially unraveled,²⁹ strand invasion by other collagen molecules orcollagen analogs have not been previously reported.

Under transmission electron microscopy, collagen fibers exhibitcharacteristic banding patterns that indicate the structural integrityof the collagen molecules and their assembly.³⁰ The banding patternsalso provide approximate position markers along the length of collagenmolecules. The binding event between type I collagen fibers and CMPconjugated gold NPs was investigated to see if strand invasion occurswithout destroying the native structure of collagen fibers. Antibodypassivated gold NPs have been used successfully to identify specifictypes of collagen fibers in tissue samples.³¹

A series of three CMPs was synthesized, each with a single cysteine atthe N terminus (Table 2, peptides 1′˜3′) using conventionalFmoc-mediated solid phase peptide coupling methods.

TABLE 2 CD Melting Transition Temperatures of CMP Derivatives CompoundSequence SEQ ID NO: Tm 1 Cys-(Pro-Hyp-Gly)3 20 — 2 Cys-(Pro-Hyp-Gly)5 2121° C. 3 Cys-(Pro-Hyp-Gly)7 22 39° C.  4a Cys- 44 — randomPro7Hyp7Gly7^(a)Cys-GPGP*PP*PPGPPP*GP*P*PP*GP*GG (P* = Hyp)(SEQ ID NO: 44)A fourth peptide, peptide 4′, with an amino acid composition identicalto that of peptide 3′ but with a scrambled amino acid sequence that isunable to support a triple helical structure was also prepared. Themelting transition temperatures (Table 3) of these compounds determinedby circular dichroism (CD) spectrometry was consistent with those ofother CMP analogs reported previously.^(32,33) The citrate reductionmethod was used to prepare relatively monodisperse gold NPs of diameter13.2±3.5 nm.³⁸ Incubating the gold NPs and Cys-CMPs (1′˜4′) at roomtemperature produced CMP functionalized gold NPs (Table 3; NP-X,X=1′˜4′).

TABLE 3 Properties of CMP functionalized Au NPs. Calculated MeasuredPeptide Particle Size Peptide layer layer Number of by DLS thicknessthickness peptides per NP NP-1 17.2 ± 3.7 nm  2.0 nm 2.6^(a) (2.8)^(b)582 ± 32 nm NP-2 20.5 ± 3.3 nm 3.65 nm 4.3 (4.7) 444 ± 18 nm NP-3 26.9 ±4.6 nm 6.85 nm 6.0 (6.6) 399 ± 21 nm NP-4 16.2 ± 3.1 nm  1.5 nm — 524 ±59 ^(a)CMP triple helix, ^(b)poly(proline)-II helix

The melting temperatures of CMPs bound to the NP surfaces could not bedetermined because circular dichroism measurements were hampered bylight scattering from the NPs; calorimetric analysis was unsuccessfuldue to a trace amount of CMPs present on the NP surfaces. Thermalmelting curves as determined by ellipicity of CMPs is shown in FIG. 14.The thickness of the peptide layer on the gold NP surface was determinedby dynamic light scattering (DLS) (FIG. 15). The peptide layerthicknesses of NP-1, NP-2 and NP-3 were 2.0 nm, 3.65 nm and 6.85 nm,respectively (Table 3). These values were commensurate with theestimated lengths of the corresponding CMPs in helical conformation.³⁵In contrast, the peptide layer thickness of NP-4 was only 1.5 nm,indicating that this peptide was either a random coil or had adistinctively different secondary structure that tended to lie flat onthe particle surface.

The average number of CMPs on the NPs was determined by measuring theconcentrations of free CMPs remaining in solution after the removal ofthe NPs from a series of incubation mixtures with varying CMP/NP ratios(FIG. 16 and Table 3).³⁶ The numbers of CMPs immobilized on the NPsurfaces were 582, 444, and 399 for NP-1, NP-2, and NP-3, respectively.These values correspond to the effective footprint areas (per singlechain) of 94 Å², 123 Å², and 137 Å², which are comparable to thecross-sectional area of the CMP triple helix, 80 Å².³⁵ The peptide layeris likely composed of laterally packed single stranded CMPs. This mayhave been caused by surface anchoring of the chain ends that prohibitedthe staggered arrangement of the peptide chains required for theformation of triple helix.

The DLS and average number of peptides per NP data are in agreement withCMPs standing upright on the NP surfaces in an extended conformationthat resembles the poly(proline)-II helix. In negatively stainedtransmission electron microscopy micrographs, the peptides appear as adense white layer covering the NP surface (FIGS. 17A-17D). All CMPfunctionalized NPs were able to assemble into a pseudo-hexagonallattice, indicating that the CMPs fully passivated the NP surfaces andprevented the NPs from aggregating.

The CMP conjugated NPs were highly stable in aqueous solution. No signof aggregation was detected in buffer solutions of up to 5 M NaCl orwithin a pH range from 0 to 14 (FIGS. 18A and 18B). All NPs can befreeze-dried and resuspended without aggregation of the particles.Peptides usually make poor passivating layers for NPs and even peptidesthat are carefully designed to disperse NPs did not protect NPs undersuch wide range of conditions.³⁶ CMP triple helices consist of threeparallel peptide chains; hence the association of peptides withanti-parallel alignment from two different NPs is unlikely. It is likelythat the high Hyp content and extended conformation of the peptides, asshown by DLS, caused the CMPs to mimic the behavior of poly(ethyleneglycol) brushes on surfaces and prevent the NPs from aggregating inaqueous solution.

Type I collagen fibers were prepared by adjusting the pH and ionicstrength of an acid soluble type I collagen solution.³⁷ In forming afiber, collagen molecules aligned head to tail in the direction of thelong axis in overlapping rows with a gap between the molecules withineach row. Accumulation of staining agents (uranyl acetate) in these gapregions produced dark bands that repeated every 67 nm along the lengthof the fiber. Compound 3 had the highest melting temperature among thethree CMPs, suggesting that it likely makes the most stable adduct withthe collagen fiber. Therefore, NP-3 binding to reconstituted collagenfibers was characterized.

When NP-3 or NP-4 was allowed to bind to type I collagen fibers byincubating in a phosphate buffer at 25° C., transmission electronmicroscopy studies revealed drastically contrasting results. NP-4exhibited little binding to the collagen fibers, whereas NP-3 was highlyattracted to collagen fibers (FIG. 19A, 19B, and 19C). Moreover, NP-3seemed to bind to defined locations within the gap regions along thefiber axis (FIG. 19C). Due to the large particle size and limitations oftransmission electron microscopy resolution, the exact locations ofthese binding sites could not be determined. Twice as many NP-3particles per unit area was found in the gap regions (dark bands) thanon the overlap regions (light bands) of the collagen fibers. Thisdifference was abolished at incubation temperatures above 40° C. (FIG.20).

These results indicated that CMP-functionalized NPs bind to the collagenfiber in its native state, possibly at specific locations withincollagen molecules, and that the CMP's propensity to form a triple-helixwas critical in the binding process. Collagen fibers exhibited clearbanding morphology even after incubating with NPs, suggesting that theyremained intact. This was not surprising since the incubationtemperatures in these experiments was kept below the melting temperatureof collagen fibers (65.1° C.).³⁸ Recent studies using calorimetry andisothermal circular dichroism spectroscopy demonstrated that type Icollagen molecules are unstable at body temperature.³⁹ Miles and Baileyidentified three thermally unstable domains in type I collagen that lackhelix stabilizing Hyps. Interestingly, all three domains are locatedwithin the gap region of the collagen fiber.²⁹ Without wishing to bebound by theory, it is likely that accumulation of NPs in this gapregion indicates a preferential CMP interaction with the thermallylabile domains of collagen molecules (FIG. 21). This selectivity inbinding is not apparent at higher temperatures since additional thermalenergy makes other regions of the molecules unstable and susceptible toinvasion by CMPs.

The NP labeling technique may be used to identify structuralabnormalities in collagen fibers that are related to debilitating humandiseases.¹⁵ Strand invasion or similar forms of strand association bycollagen and collagen derivatives may uncover the behaviors offibril-associated collagens (type IX and XII) and other proteins thatincorporate collagen-like sequences.

Example 5 CMP-Containing Hydrogels are Useful for Cartilage Repair

The discovery that collagen mimetic peptides can be used to modifiedcollagen compositions provides for the development of a variety oftherapeutic compositions featuring such peptides. For example, collagenmimetic peptides may be used to produce improved biocompatible tissuescaffolds that contain CMP conjugated to biochemically inert polymers.Such scaffolds retain collagens and other extracellular matrixcomponents secreted by the cells cultured within them. Thus, tissuescaffolds containing CMP reproduce a microenvironment that more closelyresembles the cell's natural environment than conventional scaffolds(FIG. 22).

Example 6 Acryloyl-PEG-CMP is a Photopolymerizable CMP Derivative

The CMP, (Pro-Hyp-Gly)₇-Tyr (SEQ ID NO: 18), was synthesized at greaterthan 99% purity. Purity was analyzed using MALDI-TOF spectrometry.(ProHypGly)₇-Tyr (SEQ ID NO: 18) was selected because the(ProHypGly)₇-CMP (SEQ ID NO: 40) unit was shown, as described above, tobind to collagen fiber at physiological temperatures. Tyr was added tothe peptide to facilitate the accurate measurement of CMP concentrationby UV-Vis spectrophotometer. The peptide was synthesized on a solidsupport (Wang resin), cleaved, and purified by reverse phase HPLC. Thepeptide's ability to form a collagen triple helix was confirmed usingcircular dichroism. The CD trace included a positive peak near 225 nm, acrossover around 215 nm, and a minimum at around 180 nm.

The CD melting temperature (midpoint of CD melting curve) of the(ProHypGly)₇-Tyr (SEQ ID NO: 18) CMP was determined to be 38.5° C. Thismelting temperature is slightly higher than that of the (Pro-Hyp-Gly)₇CMP (SEQ ID NO: 40), which had a melting temperature of 37° C. A similarchange in melting temperature was observed when a hydrophobicfluorescence tag was attached to the N-termini of a CMP.

Reaction of the purified (ProHypGly)₇-Tyr (SEQ ID NO: 18) CMP withacryloyl-PEG-N-hydroxysuccinimide produced acryloyl-PEG-CMP, aphotopolymerizable CMP derivative. A CMP/poly(ethylene oxide) diacrylate(PEODA) hydrogel was prepared by photopolymerizing an aqueous solutioncontaining the acryloyl-PEG-CMP and PEODA monomers (combined weightpercent: 10%) as well as a photo reactive initiator (0.05%).Unconjugated reactants were removed using ultrafiltration and gelpermeation chromatography. Purity of the final peptide-polymer conjugatewas 80% as determined using MALDI-TOF.

Example 7 A CMP/PEODA Hydrogel Retained Added Collagen

To determine whether a CMP/PEODA hydrogel was capable of retaining addedcollagen, varying concentrations of type I collagen were added to theinitial CMP/PEODA polymer solution. The solution was then exposed to UVradiation to produce a hydrogel and the hydrogel was incubated for oneweek in PBS buffer. The incubation in PBS provided an opportunity forthe collagen to leach out of the hydrogel. Following the incubation inPBS buffer, the hydroxyproline content of the hydrogel was assayed.Hydroxyproline content is used to measure the presence of collagen.Hydroxyproline content in the CMP containing hydrogel was significantlygreater than that observed in control gels that did not contain CMP(FIG. 23). The amount of collagen retained by the CMP/PEODA hydrogelreached saturation when the gel was incubated with 0.2 mg/mL ofcollagen. The amount of collagen retained did not increase when 0.5mg/mL of collagen was added to the CMP/PEODA hydrogels.

Example 8 CMP Containing Hydrogels have Increased Water Content

Water content of a hydrogel is closely correlated to crosslinkingdensity and mesh size of the scaffold. The water content of hydrogelswith and without CMP was determined following a twenty-four hourequilibration in chondrocyte medium. The wet and dry weights of gelscrosslinked with various concentrations of collagen mimetic peptides wasmeasured and the water content of each hydrogel was determined.Hydrogels crosslinked to CMP contained 10% more water than hydrogelsthat did not contain CMP (Table 4).

TABLE 4 Water content of hydrogels as a function of CMP concentrationPercentage of CMP (%) Water Content (Q, %) S.D. 0 82.963 0.11401 0.190.355 0.22550 0.5 90.444 0.30966 1 90.608 3.3092 2 90.844 0.60216Water content was significantly higher in the CMP/PEODA hydrogelsrelative to the PEODA hydrogel. This was not surprising because a singlePEG-CMP molecule is crosslinked to each PEODA molecule the hydrogelcontaining CMP has a lower crosslinking density than the hydrogelwithout CMP.

Little difference in water content was observed in CMP/PEODA hydrogelscontaining greater than 0.1% of ACRL-PEG-CMP. In fact, hydrogelscontaining 0.1-2% CMP all had similar water contents (approximately 90%water). This may be a result of CMP's tendency to associate with andform triple helixes with other CMPs. Since the triple helix bringstogether three separate chains to form a single complex, theseassociation effectively crosslink the hydrogel and may affect theswelling characteristics of the gel.

Example 9 A CMP/PEODA Hydrogel Supported Cell Survival

To determine whether the peptide-polymer conjugate was capable ofsupporting cell survival, chondrocytes were harvested and encapsulatedin control, 1%, and 2% CMP/PEODA hydrogels. The CMP containing hydrogelsuccessfully supported cell survival as shown by a cell viability assay(FIG. 24). Moreover, cell viability was homogenous throughout theCMP/PEODA gels (FIG. 24).

Example 10 A CMP/PEODA Hydrogel Retained Cell-Secreted Collagen

Cartilage is mainly comprised of collagen and proteoglycans. Collagen isa good marker of the cultured chondrocyte's biosynthetic capabilitybecause collagen is the most abundant extracellular matrix component incartilage. The collagen produced by chondrocytes is mostly type IIcollagen. Type II collagen is also expected to physically interact withCMP. To determine whether the collagen secreted from the encapsulatedcells bound to the CMP/PEODA hydrogel, collagen content present in thehydrogel was assayed following one week of chondrocyte culture. Collagencontent was 37% higher in the 2% CMP/PEODA hydrogel than in the 1%CMP/PEODA hydrogel. Total collagen production was CMP dose dependent andfor the 2% CMP/PEODA gel, the ratio of collagen content in the two gel(PEODA:2% CMP/PEODA=1:2) was comparable to that determined from themodel collagen retention experiment. Only negligible amounts of collagenwere detected in acellular hydrogels.

Example 11 Cells Grown in CMP/PEODA Hydrogel Secreted ExtracellularMatrix Components that were Retained by the Hydrogel

Production of extracellular matrix components, such as collagen andglycosaminoglycan, is indicative not only of cell viability, but also offunctionality. Histological evaluation was used to determine whetherencapsulated chondrocyte cells were distributed evenly throughout of thehydrogel and whether the cells were secreting extracellular matrixcomponents (FIG. 17). To determine whether chondrocytes were producingglycosaminoglycan (GAG), which is a component of the extracellularmatrix, GAG content in the hydrogel was assayed following two weeks ofculture. GAG is a hydrophilic polysaccharide unit attached toproteoglycans that, in combination with collagen, is a major constituentof cartilage extracellular matrix. GAG synthesis in the 2% CMP/PEODAhydrogel was significantly greater than that in thel % CMP/PEODAhydrogel and control PEG gel, by 105% and 87%, respectively. However,there was no significant difference in GAG production between thecontrol and 1% CMP/PEODA hydrogel.

The total GAG assay indicated that the GAG production was not influencedby the presence of 1% PEODA-CMP in the hydrogel, even though thePEODA-CMP hydrogel had significantly higher water content than PEODA.Typically, increasing the water content of a hydrogel increases thetissue remodeling ability at the expense of mechanical properties. Themechanical strength of the hydrogel is weakened but due to higher watercontent, cell can take up nutrients easily and produce high levels ofECM. This was not observed in the CMP/PEODA hydrogel. Although 1% and 2%CMP/PEODA had similar water content, the GAG content of 2% CMP/PEODA was1.9 times higher than that of the 1% CMP/PEODA. This indicates that theincrease in GAG production was not the result of an increase in watercontent.

Proteoglycan deposition was also examined using safranin-O staining. Theintensity of safranin-O staining was highest in the 2% CMP/PEODA gels,and was next highest in the 1% CMP/PEODA hydrogel. Total collagenstaining in the CMP/PEODA hydrogel was also examined using Masson'sTrichrome stain. The CMP/PEODA hydrogel was more intensely stained thanthe PEODA gel. No difference in staining intensity was observed inhydrogel's containing varying CMP concentrations. No staining wasobserved in a control acellular CMP/PEODA hydrogel. Immunohistochemicalstaining for collagen type II also showed strong positive staining in 1%and 2% CMP/PEODA hydrogels (FIG. 26).

Chondrocytes produce the matrix material and the collagen fibers presentin bone. As the chondrocytes secrete matrix material around them, theybecome walled off into small chambers or lacunae. The chondrocytesembedded in the hydrogel had a spherical morphology and were present inisolated pockets resembling lacunae. Immediately surrounding the lacunaewas a territorial matrix where newly synthesized ECM products werepresent. Heavy staining for collagen (both total collagen staining andtype II collagen immunostaining) was observed in the territorial regionsof the CMP/PEODA hydrogel indicating that the cell-secreted collagenremains in the chondrocytes microenvironment. It is likely that the highcollagen concentration around the chondrocytes simulated a more naturalmicroenviroment for the cells and enhanced its productivity of GAG andpossibly other ECM molecules. GAG immunostaining was not confined toareas immediately surrounding the cell, suggesting that GAGs are capableof diffusing through the hydrogel. This is not surprising because CMPhas no affinity for GAGs. There was enhanced matrix production inCMP/PEODA hydrogel. This may be the result of either collagen retentionor an upregulation in gene expression.

Collagen synthesis in the 1% and 2% CMP/PEODA hydrogels was 47% and 103%respectively. Chondrocytes in CMP-containing hydrogels synthesized morecollagen than chondrocytes in control hydrogels lacking CMP. Tissueformation was greater in CMP/PEODA hydrogels. Results of the biochemicalassays of chondrocyte-encapsulated hydrogel were consistent with thehistological findings (FIG. 27). DNA contents were similar in allsamples. The high initial density of chondrocytes prevented theirproliferation in the hydrogel.

In sum, the results reported herein indicate that CMP/PEODA hydrogelprovides an effective scaffold for cartilage regeneration that issuperior to existing tissue scaffolds. Given these results, CMP/PEODAhydrogels are useful for the repair of damaged cartilage, and areparticularly useful for applications involving the repair of articularcartilage.

Example 12 Reduction of Thrombus Deposition by Applying CMP Derivatives

Angioplasty denudes the vessel wall of the endothelial layer. Vesselhealing is often accompanied by the overproliferation of smooth muscleunder the endothelial layer. This process can narrow or even block bloodvessels causing thrombosis formation⁴⁰. Methods for preventing intimalhyperplasia and thrombosis subsequent to angioplasty are urgentlyrequired. The present invention provides a method of treating bloodvessel injury using CMP to deliver anti-thrombotics. CMP may beconjugated to virtually any anti-thrombotic agent known in the art. Suchagents include aspirin, thienopyridine, heparin, saratin (a 12,000 Darecombinant protein isolated from the saliva of the medicinal leechHirudo medicinalis), hirudin (a 65 amino-acid polypeptide), pegylatedhirudin, and unfractionated heparin (UH).

Desirably CMP-anti-thrombotic conjugates bind collagen (type III)⁴⁹ andrepel cell attachment. Saratin-CMPs, hirudin-CMPs, and PEG-CMPs areproduced using standard methods and purified using HPLC. Purifiedconjugates are then characterized using MALDI-TOF and CD analysis asdescribed herein. The activity of the CMP conjugates will be assessed inanti-platelet activation and anti-thrombin formation assays.Specifically, plasma levels of β-thromboglobulin (β-TG) andthrombin-antithrombin complexes (T-AT) will be determined usingimmunoenzymoassays⁵⁰. Optionally, the activity of CMP-anti-thromboticconjugates are assessed in animal experiments.

In particular embodiments, the following CMP conjugates are employed asanti-thrombotics.

Materials

Fmoc-amino acids were purchased from Advanced ChemTech (Louisville, Ky.)and Fmoc-Gly-Wang resin was purchased from Novabiochem (La Jolla,Calif.). Acid soluble, type I bovine collagen was purchased from Sigma(St. Louis, Mo.) and mPEG₂₀₀₀-Gly₃-(ProHypGly)₇ (SEQ ID NO: 6) (peptide5) was purchased from Genscript Corporation (Piscataway, N.J.). Allother chemicals were purchased from Sigma-Aldrich and used withoutfurther purification. Fibroblast (CRL-1502) cells and breast epithelialcells (MCF-7) were from ATCC (Manassas, Va.). Dulbecco's modifiedeagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM)were purchased from Invitrogen Corporation (Carlsbad, Calif.). TEMimages were acquired on a Philips 420 EM using holey carbon grids fromTed Pella Inc (Redding, Calif.). UV-Vis spectra were measured in a Cary50 Bio spectrophotometer. Thermal melting transition measurements wereperformed in a Jasco 710 spectropolarimeter. DLS data was acquired on aMalvern Zetasizer 3000.

Synthesis and Purification of Collagen Mimetic Peptides (CMPs)

The peptides were synthesized by condensation of the correspondingFmoc-amino acids (Fmoc-Gly-OH, Fmoc-Hyp-OH, and Fmoc-Pro-OH) and5-carboxyfluorescein (5CF) on a solid support (Wang resin). Both manualand automated systems (Applied Biosystems 431A Peptide Synthesizer) wereused to prepare peptides 1-4. Four-fold molar excess of the above aminoacids was used in a typical coupling reaction. Fmoc-deprotection wasaccomplished by treatment with 20% (v/v) piperidine in dimethylformamide (DMF) for 1 hour. The coupling was achieved by treatment with2-(1H-benzo-triazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) in N,N-diisopropylethylamine (DIPEA). All thecoupling reactions were completed within 3-4 hours and were monitored byninhydrin or chloranil tests.

The CMPs were cleaved from the resins by treatment withwater/triisopropylsilane/trifluoroacetic acid (2.5:2.5:95) for 3 hours.The crude CMPs were precipitated with cold ether and dried.Reverse-phase HPLC purification was performed on a Varian Polaris 210series Liquid Chromatograph with a Vydac C-18 reversed-phase column at aflow rate of 1 ml/min. The purified peptides were analyzed by amatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometer (Kompact SEQ, KRATOS or Voyager DE-STR, AppliedBiosystems). (M+H)⁺=(peptide 1, 2690.7 Da; peptide 2, 3220.23 Da;peptide 3, 3221.65 Da; peptide 4, 2296.0 Da; peptide 5, 4305.1 Da).

Circular Dichroism Measurements

Circular dichroism (CD) spectra were recorded on JASCO 710 spectrometerequipped with JASCO PTC-348 WI temperature controller and Hellma cell(400 μL, 0.1 mm pathlength). The thermal melting curves were obtained bymeasuring the molar ellipticity at 225 nm with 1° C./min heating rate.All samples (57 μM in 50 mM acetic acid) were stored at 4° C. for 24hours before the CD measurement.

CMP-Collagen Film Affinity Study

To each well of a 96-well culture plate was added 200 μl of saturatedcollagen (acid soluble, type I bovine) solution in 0.5 M acetic acid.The culture plate was air-dried to form transparent collagen films. Thefilms were neutralized with 0.01 M potassium phosphate buffer (pH 7.4),and washed with distilled water. To the collagen-coated wells were added40 μl of 0.01 M potassium phosphate solutions (pH 7.4) containing 50 μMof either CF (5-carboxyfluorescein), FITC-Dextran, and peptides 2, 3, or4, which were pre-equilibrated at 25° C. or 80° C. After 3 hours ofincubation at room temperature, the wells were washed with 0.01 Mpotassium phosphate buffer solution (pH 7.4) and observed byfluorescence microscope (Eclips ME 600, Nikon Corp). The totalfluorescence intensity was acquired from the fluorescence micrographsusing Meta Imaging Series V4.5, (Universal Imaging Corporation,Dowingtown Pa.). Average values of four independent experiments arereported.

Helical Content Estimation of the Modified Collagen Films.

Collagen (type I, bovine) film was treated with either peptide 2solution or blank buffer solution as described before. After 3 hr ofincubation at room temperature, the wells were washed with 0.01 Mpotassium phosphate buffer solution (pH 7.4) and deionized water. Allsamples were dissolved in 50 mM acetic acid to a concentration of 1.80μM or 3.60 μM and stored at 4° C. for 24 hours before the CDmeasurement. Average values of four independent experiments are reported(FIG. 4).

TEM Analysis of the Modified Collagen Fibers

Collagen fibers were prepared by dialysis of collagen in 0.01M aceticacid solution against dilute NaCl solution. Peptide 4 in potassiumphosphate buffer (400 μl, 500 pH 7.4) was added to the collagen fibersin the PBS buffer (1′ ml, 1 mg/ml, pH 7.4). The mixture was incubatedfor 3 hours and excess peptide 4 was removed by repeated centrifugation.The collagen fibers were resuspended in deionized water and transferredto a TEM grid. Uranyl acetate was used to stain the collagen fiber.(FIG. 6)

Cell (Human Fibroblasts and Breast Epithelial Cells) Adhesion Study

To the collagen film (prepared as above) was added 10 μl of peptide 5solution (2 mM in pH 7.4 potassium phosphate buffer). The film wasallowed to dry and excess materials were removed by washing withpotassium phosphate buffer (pH 7.4, 50 mM) and culture medium (DMEM orIMDM). Cells were added to the culture well and incubated at roomtemperature for 30 minutes. Human fibroblasts (CRL-1502) were seeded at4.5×10⁵ cell/ml cell density in DMEM and breast epithelial cells (MCF-7)at 5.6×10⁵ cells/ml density in cell culture media (IMDM). Thenonattached cells were removed by rinsing the well three times withgrowth medium and PBS buffer. The remaining cells were incubated at 37°C. (5% CO₂) for three days. The growth medium was exchanged after 48hours.

Synthesis of Gold Nanoparticles.

Gold nanoparticles were synthesized by citrate reduction methodaccording using standard methods.⁴¹ Briefly, tri-sodium citrate solution(25 mL, 38.8 mM) was quickly added to a refluxing aqueous solution ofHAuCl₄ (250 mL, 1 mM) with vigorous stirring. The color of the solutionchanged from pale yellow to deep red after adding the citrate solution.The solution was refluxed for an additional 15 minutes, cooled, andfiltered through a glass filter.

Synthesis and Purification of CMPs.

CMP 1′˜4′ were synthesized and purified as described above.⁴²

Thermal Melting Curves of CMPs.

The thermal melting curves were obtained by measuring the ellipticity ofpeptide in deionized (DI) water (0.5˜2 mM) at 225 nm with 0.1° C./minheating rate. All samples were stored at 4° C. for 24 hours before themeasurement. (FIG. 15)

Surface Functionalization of Gold NP with Cys-CMPs.

Purified Cys-CMP (peptides 1′˜4′) (0.5 mL, 500 μM) was added to goldnanoparticle solution (5 mL, 17 nM), and the reaction mixture wasincubated at room temperature for twenty-four hours. Excess CMPs wereremoved by repeated centrifugation and washing in deionized water. TheDLS results of NP-Xs are shown in FIG. 16.

Stability of NP-Xs.

The aggregation parameter (AP) is defined as follows: AP=(A−A₀)/A₀,where A is the integrated absorbance between 600 and 700 nm of a sampleat a given condition, and A₀ is the integrated absorbance of a fullydispersed NP solution.³

Regeneration of Type I Collagen Fibers.

Collagen fibers were regenerated by mixing 0.5 mL of acid soluble type Icollagen (1 mg/mL) in 50 mM acetic acid solution with 1 mL of sodiumphosphate buffer (10 mM, pH 8.25). The mixture was incubated at roomtemperature for 12 hours.

Tem Analysis of NP-3 Decorated Collagen Fibers.

A drop (8 μL) of type I collagen solution (0.3 mg/mL in PBS containing1% BSA) was added to a holey carbon TEM grid. NP-3 solution (3 nM in PBScontaining 1% BSA) was added to the grid and incubated for 5 minutes atroom temperature or at 40° C., followed by washing with deionized water.The collagen fibers were stained with uranyl acetate (1%).

Measurement of Number of Peptides Per NP (Passivation Number).

Seven 10 μM Cys-CMP solutions with varying concentrations ofcitrate-stabilized gold NP (2˜14 nM) were prepared. The mixtures wereincubated for 2 hours at 25° C. and the gold NPs were removed bycentrifugation. The concentrations of free peptides remaining in thesupernatant were determined from the UV-Vis absorbance at 215 nm withcitrate ion background subtraction. The number of peptides per Au NP wascalculated from the slope of the linear fit of the data points. (FIG.17)

Synthesis and Purification of Collagen Mimetic Peptide (CMP)

(Pro-Hyp-Gly)₇-Tyr (SEQ ID NO: 18), CMP, was synthesized by Fmocmediated solid phase peptide coupling methods starting from a tyrosinepreloaded Wang resin. High performance liquid chromatography (HPLC: C18Vydac column) was used to purify the peptide. The peptide product waslyophilized and stored at −20° C. The molecular weight of the productwas confirmed by Matrix-assisted laser-desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS, Voyager DE-STR, AppliedBiosystems).

Triple-helical conformation of the CMP was confirmed by circulardichroism (CD) spectroscopy. Peptide samples (250 or 500 μM) wereprepared in distilled water and incubated at 4° C. overnight before theCD measurement. CD was recorded between 180 and 280 nm on a JASCO 710 CDspectrometer at various temperatures under a nitrogen flow. Eachmeasurement had static equilibrium time of 30 minutes.

Synthesis and Purification of Acryloly-PEG-Peptide

One milligram (4.9 μM) of (Pro-Hyp-Gly)₇-Tyr (SEQ ID NO: 18) wasdissolved in 1 ml of 0.05 M sodium bicarbonate solution. Acryloyl(ACRL)-PEG-peptide was prepared as previously described⁴⁴. Briefly,acryloyl-PEG-N-hydroxysuccinimide (ACRL-PEG-NHS, Nektar, 3.4 mg, 10 μM)was dissolved in 200 μL of 0.05 M sodium bicarbonate solution and theresulting solution was added drop-wise to the CMP solution. The solutionwas shaken on an orbital shaker for 2 hours at room temperature.Ultrafilration (Millipore, molecular weight cut off=3500) was used toremove unreacted CMP and ACRL-PEG-NHS, and the solution containing thepure product was lyophilized. The dried powder was dissolved indeionized water and run through a size exclusion column Sephadex® G-25(Pharmacia, motive phase: deionized water) to remove small moleculeimpurities. Elution fractions containing the target product werecombined and lyophilized. MALDI-TOF was used to confirm the molecularweight of the target product. Product was stored at −20° C. and usedwithin a week.

Collagen Encapsulation in Hydrogels

Type I collagen in 0.1 N acetic acid (Sigma) was neutralized andresuspended in macromer solutions (0.05 wt % initiator (D-2959, Ciba), 8wt % PEODA (molecular weight: 3400, Nektar) and 2% ofACRL-PEG-(Pro-Hyp-Gly)₇-Tyr) (SEQ ID NO: 45) at a concentration of 0.2or 0.5 mg/ml. Control samples composed of 10 wt % PEODA were alsoprepared. Ultraviolet lamp (EXFO Acticure 4000; wavelength: 365 nm;intensity 5 mW/cm²; 5 min. exposure time) was used to photopolymerize100 μL of the macromer collagen solution mixture. The polymerized gelconstructs were transferred to 12 well tissue culture plates with eachwell containing 2 ml PBS buffer. The culture plate was incubated at 37°C. in 5% CO₂ atmosphere, and PBS buffer was replaced every 2-3 days forone-week period.

Collagen Assay

After one week of incubation, the wet weight and dry weight ofcollagen-encapsulated hydrogel was determined by measurement of thehydrogel before and after 48 hours of lyophilization, respectively. Adry construct was digested in 900 ml of papain solution (250 μg/mlpapain type II (Worthington Biomedical Corporation, Lakewood, N.J.), 100mM phosphate buffer, 10 mM EDTA, 10 mM cysteine at pH 6.3) for overnightat 60° C. The digests were then centrifuged at 10,000 rpm for 5 minutes,and supernatants were taken.

Total collagen content of the gel was estimated from the hydroxyprolineassay conducted with papain-digested solution after overnight hydrolysisreaction with 6N hydrochloric acid at 115° C.⁴⁵. Hydrolyzed samples werereacted with p-dimethylamino benzaldehyde and chloramines-T hydrate⁴⁶.Absorbance was measured at 550 nm on a UV-V is spectrophotometer.Standard curve was generated using pure trans-4-hydroxy-L-proline(Sigma-Aldrich). Collagen content of the blank gel (no collagen) wasdetermined and subtracted in the calculation of the total collagencontent.

Chondrocyte Isolation and Encapsulation

Chondrocytes were isolated from the femoral patellar groove and femoralcondyles of three 5- to 8-week-old calves. The articular cartilage wasexcised under aseptic conditions and digested overnight at 37° C. in0.2% Collagenase type II (Gibco) and 5% fetal bovine serum (FBS, Gibco,Carlsbad, Calif.) in Dulbecco's modified Eagle medium (DMEM, Gibco). Thedigested suspension was filtered through a cell strainer (FisherScientific Co.) and centrifuged at 1500 rpm for 10 minutes. Thesupernatant was removed and the pellet was washed and resuspended in PBSbuffer (Gibco) supplemented with 1% penicillin-streptomycin (Gibco). Thepellet was washed twice and resuspended in medium.

Harvested cells were resuspended in the macromer solutions at aconcentration of 20×10⁶ per ml. For example, macromer solutions for 2 wt% CMP-PEG gel contained initiator (0.05 wt %; D-2959, Ciba), PEODA (8 wt%; Nektar), ACRL-PEG-(Pro-Hyp-Gly)₇-Tyr (SEQ ID NO: 45) (2 wt %) and PBSbuffer (89.95 wt %). Each gel-construct was prepared in 100 μLcell-macromer solution and photopolymerized as described previously. Theconstruct was transferred to a well of 12-well culture plate containing2 ml of culture medium. The medium was composed of 10 mM HEPES (Gibco,Carlsbad, Calif.), 0.1 mM NEAA (Gibco, Carlsbad, Calif.), 0.4 mM proline(Sigma), 50 mg/L Vitamin C (Sigma), 10% FBS, and 1%penicillin-streptomycin in high glucose DMEM. Constructs were culturedat 37° C. in 5% CO₂ atmosphere and medium was replaced every 2-3 days.

Cell Viability Test

The viability of hydrogel encapsulated cells was determined after 2weeks of culture. The medium was discarded and the hydrogel matriceswere washed twice with phosphate buffer solution (PBS, Gibco, Carlsbad,Calif.). Cell viability was assessed based on the integrity of cellularmembrane using Live/Dead Viability/Cytotoxicity Kit (Molecular Probes,Eugene, Oreg.) that contains calcein-AM (“Live”) dye and ethidiumhomodimer-1 (“Dead”) dye. Dye solution was made with 0.5 μL ofcalcein-AM dye and 2 μl of ethidium homodimer-1 dye in 1 mL DMEM. Aslice of the cell containing matrix was incubated in 500 μL of the“Live/Dead” dye solution for 30 minutes. Fluorescence microscopy wasperformed using a fluorescein optical filter (485±10 nm) for calcein-AMand a rhodamine optical filter (530±12.5 nm) for ethidium homodimer-1.

Histology and Immunohistochemistry

After two weeks of culture, cell containing and control matrices werefixed overnight in 10% formalin solution and stored in 70% ethanol. Thefixed cell containing matrix was embedded in paraffin, sectioned andstained with Safranin-O/fast green or with Masson's trichrome stain.Acellular controls of the CMP/PEODA hydrogel were also stained followingthe same procedure to measure any background signals.Immunohistochemistry was performed using rabbit polyclonal antibody tocollagen type II (Research Diagnostics) and a Histostain-SP kit (Zymed).

Water Content and Biochemical Assays

Water content of the cell-encapsulated hydrogels was calculated by thefollowing equation:Water content (%)=[(W _(w) −W _(d))/W _(w)]×100  (1)where W_(w) is wet weight of the hydrogel after one day equilibration inwater, and W_(d), dry Weight of the hydrogel.

Cell containing and control matrices were digested with Papainovernight. The digested samples were used for both DNA andglucosaminoglycan (GAG) measurements. Hoechst 33258 dye from MolecularProbes (Eugene, Oreg.) was used for the DNA assay⁴⁷. The solution (0.1μg/ml) was prepared in 1×THE buffer (10 mM Tris, 1 mM EDTA, 0.2 M NaCl,pH 7.4). Calf Thymus DNA standards were prepared with 0 to 100 μg/mlDNA. Papain-digested samples (30 or 60 μL) or standards were mixed withthe prepared dye solution. DNA content was measured with a fluorometer(365 nm excitation and 458 nm emission) and calculated from the calfthymus DNA standard curve. The GAG was measured using thedimethylmethylene blue (DMMB) dye⁴⁸. A standard curve was created withchondroitin sulfate C (shark cartilage extract, Sigma). Absorbance wasmeasured at 525 nm on a UV-Vis spectrophotometer. Collagen assays werealso performed as described above. Collagen contents in the acellularCMP hydrogels were also measured for the purpose of backgroundsubtraction. GAG and collagen contents from the biochemical assays werenormalized by the DNA content. The value is presented as mean±standarddeviation. Statistical significance was determined by unpaired Student ttest and set as p<0.05.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

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1. A method for modifying collagen, the method comprising contactingcollagen with a collagen mimetic peptide consisting of Z-[X-Y-Gly]₂₋₂₀repeat unit (SEQ ID NO:1), wherein Z is any amino acid, X is proline ormodified proline, Y is proline or modified proline, under conditionsthat provide for a physical interaction between the collagen and thecollagen mimetic peptide.
 2. The method of claim 1, wherein Z, X, and/orY is modified proline.
 3. The method of claim 1, wherein the collagenmimetic peptide is conjugated to an antibiotic, a cell adhesionmolecule, a contrast agent, a detectable label, a growth factor, acomponent of the extracellular matrix, an anti-inflammatory agent, apolymer, polyethylene glycol (PEG), or a small molecule.
 4. A method formodifying collagen, the method comprising contacting collagen with acollagen mimetic peptide consisting of an amino acid sequence selectedfrom the group consisting of: Gly₃-(ProHypGly)₆, (SEQ ID NO:13)Gly₃-(ProHypGly)₇, (SEQ ID NO:14) Gly₃-(ProHypGly)₈, (SEQ ID NO:15)Gly₃-(ProHypGly)₉, (SEQ ID NO:16) (ProHypGly)₆-Tyr, (SEQ ID NO:17)(ProHypGly)₇-Tyr, (SEQ ID NO:18) (ProHypGly)₈-Tyr, (SEQ ID NO:19)Cys-(Pro-Hyp-Gly)₃, (SEQ ID NO:20) Cys-(Pro-Hyp-Gly)₅, (SEQ ID NO:21)and Cys-(Pro-Hyp-Gly)₇ (SEQ ID NO:22) under conditions that provide fora physical interaction between the collagen and the collagen mimeticpeptide.
 5. The method of claim 2, wherein the modified proline is4-hydroxyproline or 4-fluoro proline.
 6. The method of claim 1, wherein[X-Y-Gly] is (ProHypGly), (ProProGly), or (ProFlpGly), where Hyp ishydroxyproline and Flp is 4-fluoroproline.